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Sample records for baculovirus-infected insect cells

  1. Display of a Maize cDNA library on baculovirus infected insect cells

    Directory of Open Access Journals (Sweden)

    Jones Ian M

    2008-08-01

    Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  2. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

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    Daniel Mendes Pereira Ardisson-Araújo

    Full Text Available Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3 has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV expression. Five different forms of Ba3 were assessed; (1 the full-length sequence, (2 the pro-peptide and mature region, (3 only the mature region, and the mature region fused to an (4 insect or a (5 virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect

  3. Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

    Science.gov (United States)

    Xuan, X; Maeda, K; Mikami, T; Otsuka, H

    1996-12-01

    The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

  4. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

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    Nuno Carinhas

    Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  5. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

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    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  6. Arbovirus vaccines: opportunities for the baculovirus-insect cell expression system

    NARCIS (Netherlands)

    Metz, S.W.H.; Pijlman, G.P.

    2011-01-01

    The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging

  7. Probability to produce animal vaccines in insect baculovirus ...

    African Journals Online (AJOL)

    Administrator

    2011-09-07

    Sep 7, 2011 ... The insect baculovirus expression system is a valuable tool for the production of vaccine. .... vaccine expression/delivery vehicle (Yu-Chen et al., ... baculoviruses are applied in cell-based assays for drug ... Intramuscular.

  8. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

    International Nuclear Information System (INIS)

    Jarvis, Donald L.

    2003-01-01

    The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains

  9. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    Science.gov (United States)

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

  10. MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

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    Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

    2013-06-01

    MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

  11. Pathogenesis induced by (recombinant) baculoviruses in insects

    NARCIS (Netherlands)

    Flipsen, H.

    1995-01-01

    Infection of insect larvae by a baculovirus leads to cessation of feeding and finally to the death of the larva. Under optimal conditions this process may take as little as five days during which the virus multiplies approximately a billion times and transforms 30% of the larval weight into

  12. Probability to produce animal vaccines in insect baculovirus ...

    African Journals Online (AJOL)

    The insect baculovirus expression system is a valuable tool for the production of vaccine. Many subunit vaccines have been expressed in this system. The first vaccine produced in insect cells for animal use is now in the market. In this study, we reviewed recent progress of animal's vaccine production for different expression ...

  13. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    Science.gov (United States)

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    International Nuclear Information System (INIS)

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M.

    1990-01-01

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [ 3 H]azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the [ 3 H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart

  15. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    Science.gov (United States)

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  16. A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

    Science.gov (United States)

    Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

    2011-01-01

    The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

    International Nuclear Information System (INIS)

    Ye Ling; Lin Jianguo; Sun Yuliang; Bennouna, Soumaya; Lo, Michael; Wu Qingyang; Bu Zhigao; Pulendran, Bali; Compans, Richard W.; Yang Chinglai

    2006-01-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection

  18. Development of an influenza virus vaccine using the baculovirus-insect cell expression system : implications for pandemic preparedness

    NARCIS (Netherlands)

    Cox, M.M.J.

    2009-01-01

    Key word

    Influenza, rHA, vaccine, baculovirus, insect cells, production, pandemic preparedness

    Influenza (or flu) is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world and is caused by influenza viruses. Influenza

  19. A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

    Science.gov (United States)

    Lu, M; Farrell, P J; Johnson, R; Iatrou, K

    1997-12-05

    It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant

  20. The Current Status of Baculovirus and Their Implication for Insect Pest Control

    Directory of Open Access Journals (Sweden)

    Arman Wijonarko

    2001-07-01

    Full Text Available Baculovirus have been promoted as the promising bioinsecticides for their pest control potential for more than half a century. But only a few have been successful as biological control agent, and almost none has been proven as commercial success, or widely used for large-scale insect pest control. The bioinsecticides currently represent only a small fraction of the world pesticide market. The successful of the Bt crop marked a special achievement in the bioinsecticide market growth. How about the baculoviruses? The main hurdle for baculovirus to be developed as bioinsecticide is its poor performance compare to synthetic chemical ones, include the speed of kill, and host range. It is important to understand the nature of baculovirus, and explore the possibilities to develop new way in applying the baculovirus as bioinsecticides. Key words: current status, baculovirus, insect control

  1. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    Science.gov (United States)

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The role of baculovirus apoptotic suppressors in AcMNPV-mediated translation arrest in Ld652Y cells

    International Nuclear Information System (INIS)

    Thiem, Suzanne M.; Chejanovsky, Nor

    2004-01-01

    Infecting the insect cell line IPLB-Ld652Y with the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, which correlates with the presence of the AcMNPV apoptotic suppressor, p35. In this study, we investigated the role of apoptotic suppression on AcMNPV-induced translation arrest. Infecting cells with AcMNPV bearing nonfunctional mutant p35 did not result in global translation arrest. In contrast, global translation arrest was observed in cells infected with AcMNPV in which p35 was replaced with Opiap, Cpiap, or p49, baculovirus apoptotic suppressors that block apoptosis by different mechanisms than p35. These results indicated that suppressing apoptosis triggered translation arrest in AcMNPV-infected Ld652Y cells. Experiments using the DNA synthesis inhibitor aphidicolin and temperature shift experiments, using the AcMNPV replication mutants ts8 and ts8Δp35, indicated that translation arrest initiated during the early phase of infection, but events during the late phase were required for global translation arrest. Peptide caspase inhibitors could not substitute for baculovirus apoptotic suppressors to induce translation arrest in Ld652Y cells infected with a p35-null virus. However, if the p35-null-AcMNPV also carried hrf-1, a novel baculovirus host range gene, progeny virus was produced and treatment with peptide caspase inhibitors enhanced translation of a late viral gene transcript. Together, these results indicate that translation arrest in AcMNPV-infected Ld652Y cells is due to the anti-apoptotic function of p35, but suggests that rather than simply preventing caspase activation, its activity enhances signaling to a separate translation arrest pathway, possibly by stimulating the late stages of the baculovirus infection cycle

  3. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

    International Nuclear Information System (INIS)

    Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.; Kwang, Jimmy

    2009-01-01

    White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.

  4. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

  5. Genomic support for speciation and specificity of baculoviruses

    NARCIS (Netherlands)

    Jakubowska, A.K.

    2010-01-01

    Keywords: baculovirus, insects, speciation, genomics, phylogeny, host specificity

    The Baculoviridae comprise a large family of double-stranded DNA viruses infecting
    arthropods. In this thesis two baculoviruses, Leucoma salicis nucleopolyhedrovirus
    (LesaNPV) and Agrotis

  6. Insect Cells as Hosts for Recombinat Proteins

    OpenAIRE

    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  7. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2011-05-01

    Full Text Available Abstract Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE containing the envelope gene (env of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine.

  8. Baculovirus DNA replication

    OpenAIRE

    Kool, M.

    1994-01-01

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous proteins for various applications (Luckow and Summers, 1988; Luckow, 1991). In both cases large-scale systems for the production of baculoviruses are important. Production in insect larvae is difficult t...

  9. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  10. BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

    Science.gov (United States)

    The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

  11. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

    1987-10-01

    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

  12. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

    International Nuclear Information System (INIS)

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.

    1987-01-01

    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120

  13. Effective chikungunya virus-like particle vaccine produced in insect cells.

    Directory of Open Access Journals (Sweden)

    Stefan W Metz

    Full Text Available The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

  14. Baculovirus DNA replication

    NARCIS (Netherlands)

    Kool, M.

    1994-01-01

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous

  15. Budded baculovirus particle structure revisited

    NARCIS (Netherlands)

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2015-01-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope

  16. Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells

    DEFF Research Database (Denmark)

    Blank, Simon; Seismann, Henning; Plum, Melanie

    2011-01-01

    for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules...... were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell...

  17. Budded baculovirus particle structure revisited

    NARCIS (Netherlands)

    Wang, Qiushi; Bosch, Berend Jan; Vlak, J.M.; Oers, van M.M.; Rottier, P.J.; Lent, van J.W.M.

    2016-01-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope

  18. Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

    Directory of Open Access Journals (Sweden)

    Joergensen Louise

    2010-11-01

    Full Text Available Abstract Background The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1 antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed. Methods The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE. Results All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay. Conclusions The baculovirus based insect cell

  19. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  20. Baculovirus expression vector system: An efficient tool for the ...

    African Journals Online (AJOL)

    Baculovirus expression vector system is considered one of the most successful and widely acceptable means for the production of recombinant proteins in extremely large quantities. Proper posttranslational modifications of the expressed proteins in insect cells, the usual host of baculoviruses, get them soluble, correctly ...

  1. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    Science.gov (United States)

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

  2. Error assessment in recombinant baculovirus titration: evaluation of different methods.

    Science.gov (United States)

    Roldão, António; Oliveira, Rui; Carrondo, Manuel J T; Alves, Paula M

    2009-07-01

    The success of baculovirus/insect cells system in heterologous protein expression depends on the robustness and efficiency of the production workflow. It is essential that process parameters are controlled and include as little variability as possible. The multiplicity of infection (MOI) is the most critical factor since irreproducible MOIs caused by inaccurate estimation of viral titers hinder batch consistency and process optimization. This lack of accuracy is related to intrinsic characteristics of the method such as the inability to distinguish between infectious and non-infectious baculovirus. In this study, several methods for baculovirus titration were compared. The most critical issues identified were the incubation time and cell concentration at the time of infection. These variables influence strongly the accuracy of titers and must be defined for optimal performance of the titration method. Although the standard errors of the methods varied significantly (7-36%), titers were within the same order of magnitude; thus, viral titers can be considered independent of the method of titration. A cost analysis of the baculovirus titration methods used in this study showed that the alamarblue, real time Q-PCR and plaque assays were the most expensive techniques. The remaining methods cost on average 75% less than the former methods. Based on the cost, time and error analysis undertaken in this study, the end-point dilution assay, microculture tetrazolium assay and flow cytometric assay were found to be the techniques that combine all these three main factors better. Nevertheless, it is always recommended to confirm the accuracy of the titration either by comparison with a well characterized baculovirus reference stock or by titration using two different methods and verification of the variability of results.

  3. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    Science.gov (United States)

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

  4. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    Directory of Open Access Journals (Sweden)

    Goldbach Rob W

    2011-07-01

    Full Text Available Abstract Background Chikungunya virus (CHIKV is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

  5. Baculovirus enhancins and their role in viral pathogenicity. Chapter 9

    Science.gov (United States)

    James M. Slavicek

    2012-01-01

    Baculoviruses are a large group of viruses pathogenic to arthropods, primarily insects from the order Lepidoptera and also insects in the orders Hymenoptera and Diptera. Baculoviruses have been used to control insect pests on agricultural crops and forests around the world. Efforts have been ongoing for the last two decades to develop strains of baculoviruses with...

  6. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  7. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    Science.gov (United States)

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

    Science.gov (United States)

    Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

    2017-04-01

    Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

  9. Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

    Directory of Open Access Journals (Sweden)

    A.M. Al-Sulaiman

    2017-11-01

    Full Text Available Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses’ proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD and varicella zoster glycoprotein E (VZV gE. Recombinant plasmids were generated, transfected into insect cells (SF9, and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

  10. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  11. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    Energy Technology Data Exchange (ETDEWEB)

    Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca [London Research and Development Centre, AAFC, London, ON (Canada); Kaplanoglu, Emine [London Research and Development Centre, AAFC, London, ON (Canada); Theilmann, David A. [Summerland Research and Development Centre, AAFC, Summerland, BC (Canada); Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A. [Saskatoon Research and Development Centre, AAFC, Saskatoon, SK (Canada)

    2016-12-15

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  12. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    International Nuclear Information System (INIS)

    Donly, B. Cameron; Kaplanoglu, Emine; Theilmann, David A.; Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A.

    2016-01-01

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  13. Baculovirus Genomics

    NARCIS (Netherlands)

    Oers, van M.M.; Vlak, J.M.

    2007-01-01

    Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date. The majority of these (37) are pathogenic to lepidopteran hosts; three infect sawflies

  14. DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

    Science.gov (United States)

    Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

    2003-03-01

    We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

  15. Vaccines for viral and parasitic diseases produced with baculovirus vectors

    NARCIS (Netherlands)

    Oers, van M.M.

    2006-01-01

    The baculovirus¿insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this

  16. The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

    2010-04-15

    Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

  17. Expression of recombinant proteinase 3, the autoantigen in Wegener's granulomatosis, in insect cells

    NARCIS (Netherlands)

    Van der Geld, YM; Smook, MLF; Huitema, MG; Harmsen, MC; Limburg, PC; Kallenberg, CGM

    2002-01-01

    Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four

  18. Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.

    Science.gov (United States)

    Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

    2015-10-22

    Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

  19. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  20. A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

    International Nuclear Information System (INIS)

    Brueggert, Michael; Rehm, Till; Shanker, Sreejesh; Georgescu, Julia; Holak, Tad A.

    2003-01-01

    Whereas bacterial expression systems are widely used for production of uniformly or selectively 15 N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively 15 N-labeled proteins in insect cells. The quantities of 15 N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the 15 N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression

  1. Study on Fusion Protein and Its gene in Baculovirus Specificity

    International Nuclear Information System (INIS)

    Nemr, W.A.H.

    2012-01-01

    doses around those used in Lepidoptera male sterilization technique, one of insect control program, on the susceptibility of the irradiated cells to virus infection

  2. New baculovirus recombinants expressing Pseudorabies virus (PRV) glycoproteins protect mice against lethal challenge infection.

    Science.gov (United States)

    Grabowska, Agnieszka K; Lipińska, Andrea D; Rohde, Jörg; Szewczyk, Boguslaw; Bienkowska-Szewczyk, Krystyna; Rziha, Hanns-Joachim

    2009-06-02

    The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.

  3. Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

    Directory of Open Access Journals (Sweden)

    Selander Martin

    2007-02-01

    Full Text Available Abstract Background Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. Results We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. Conclusion Widely-used adenovirus vectors for gene delivery cause a state of

  4. A novel medium for expression of proteins selectively labeled with {sup 15}N-amino acids in Spodoptera frugiperda (Sf9) insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Brueggert, Michael; Rehm, Till; Shanker, Sreejesh; Georgescu, Julia; Holak, Tad A. [Max Planck Institute for Biochemistry (Germany)], E-mail: holak.biochem@mpg.de

    2003-04-15

    Whereas bacterial expression systems are widely used for production of uniformly or selectively {sup 15}N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively {sup 15}N-labeled proteins in insect cells. The quantities of {sup 15}N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the {sup 15}N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.

  5. Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

    International Nuclear Information System (INIS)

    Yoshida, Shigeto; Kondoh, Daisuke; Arai, Eriko; Matsuoka, Hiroyuki; Seki, Chisato; Tanaka, Takao; Okada, Masaji; Ishii, Akira

    2003-01-01

    The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle

  6. Expression of tomato yellow leaf curl virus coat protein using baculovirus expression system and evaluation of its utility as a viral antigen.

    Science.gov (United States)

    Elgaied, Lamiaa; Salem, Reda; Elmenofy, Wael

    2017-08-01

    DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.

  7. Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

    Science.gov (United States)

    Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

    2017-09-15

    Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

  8. Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Santiago Haase

    2015-04-01

    Full Text Available Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

  9. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts

    International Nuclear Information System (INIS)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-01-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15 N and 13 C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor

  10. Baculoviruses and nucleosome management

    International Nuclear Information System (INIS)

    Volkman, Loy E.

    2015-01-01

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication

  11. Baculoviruses and nucleosome management

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Loy E., E-mail: lvolkman@berkeley.edu

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  12. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    International Nuclear Information System (INIS)

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P.

    1991-01-01

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [ 3 H]glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Galβ1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked α1-6 to the asparagine-linked N-acetylglucosamine

  13. An economic approach to efficient isotope labeling in insect cells using homemade {sup 15}N-, {sup 13}C- and {sup 2}H-labeled yeast extracts

    Energy Technology Data Exchange (ETDEWEB)

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan, E-mail: Stephan.Grzesiek@unibas.ch [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)

    2015-07-15

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein {sup 15}N and {sup 13}C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.

  14. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    Directory of Open Access Journals (Sweden)

    Liu Xiangtao

    2011-02-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE, and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA and indirect sandwich-ELISA (IS-ELISA showed both recombinant baculoviruses (with or without T-cell epitopes were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ. Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P P Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection.

  15. Distribution and Spectroscopy of Green Fluorescent Protein and Acyl-CoA: Cholesterol Acytransferase in Sf21 Insect Cells

    Science.gov (United States)

    Richmond, R. C.; Mahtani, H.; Lu, X.; Chang, T. Y.; Malak, H.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Acyl-CoA: cholesterol acyltransferase (ACAT) is thought to significantly participate in the pathway of cholesterol esterification that underlies the pathology of artherosclerosis. This enzyme is a membrane protein known to be preferentially bound within the endoplasmic reticulum of mammalian cells, from which location it esterifies cholesterol derived from low density lipoprotein. Cultures of insect cells were separately infected with baculovirus containing the gene for green fluroescent protein (GFP) and with baculovirus containing tandem genes for GFP and ACAT. These infected cultures expressed GFP and the fusion protein GCAT, respectively, with maximum expression occurring on the fourth day after infection. Extraction of GFP- and of GCAT-expressing cells with urea and detergent resulted in recovery of fluorescent protein in aqueous solution. Fluorescence spectra at neutral pH were identical for both GFP and GCAT extracts in aqueous solution, indicating unperturbed tertiary structure for the GFP moiety within GCAT. In a cholesterol esterification assay, GCAT demonstrated ACAT activity, but with less efficiency compared to native ACAT. It was hypothesized that the membrane protein ACAT would lead to differences in localization of GCAT compared to GFP within the respective expressing insect cells. The GFP marker directly and also within the fusion protein GCAT was accordingly used as the intracellular probe that was fluorescently analyzed by the new biophotonics technique of hyperspectral imaging. In that technique, fluorescence imaging was obtained from two dimensional arrays of cells, and regions of interest from within those images were then retrospectively analyzed for the emission spectra that comprises the image. Results of hyperspectral imaging of insect cells on day 4 postinfection showed that GCAT was preferentially localized to the cytoplasm of these cells compared to GFP. Furthermore, the emission spectra obtained for the localized GCAT displayed a peak

  16. On the role of baculovirus photolyases in DNA repair upon UV damage of occlusion bodies

    NARCIS (Netherlands)

    Biernat, M.A.; Caballero, P.; Vlak, J.M.; Oers, van M.M.

    2013-01-01

    The use of baculoviruses in insect biocontrol is hampered by their sensitivity to ultraviolet (UV) light. This irradiation induces cyclobutane pyrimidine dimers (CPDs) in DNA. CPD-photolyases repair CPDs using visible light. Plusiine baculoviruses encode photolyases, which could potentially repair

  17. Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state

    International Nuclear Information System (INIS)

    Abo, Yohichi; Hagiya, Akiko; Naganuma, Takao; Tohkairin, Yukiko; Shiomi, Kunihiro; Kajiura, Zenta; Hachimori, Akira; Uchiumi, Toshio; Nakagaki, Masao

    2004-01-01

    We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of 32 P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function

  18. Properties of Oryctes Baculovirus Isolated in Indonesia

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi

    1995-12-01

    Full Text Available An Indonesian isolate of Oryctes baculovirus was purified from infected midguts of the rhinoceros beetle (Oryctes rhinoceros by centrifugation on a 10–40% (w/v sucroese gradient. Morphological features of  nucleocapsid including a tail-like projection were very same as those previously reported. Both protein components of purified particles and restriction fragment electrophoresis profiles of viral DNA were similar to those of their isolates of Oryctes baculovirus, although there were some differences. Key words: Baculovirus oryctes, electrophoresis

  19. Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

    Science.gov (United States)

    Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

    2016-01-01

    A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

  20. The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

    Science.gov (United States)

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

    2013-10-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

  1. Genetically-engineered baculovirus pesticides and their environmental safety

    Science.gov (United States)

    H. Alan Wood; Yu Zailin

    1991-01-01

    Baculoviruses such as the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) are ecologically attractive alternatives to chemical insect pesticides but have a slow rate of control. To overcome this we have developed and are field testing an environmentally acceptable strategy which can be used for the introduction and expression of pesticide-...

  2. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution.

    Directory of Open Access Journals (Sweden)

    Toshihiro Nagamine

    Full Text Available Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV and Bombyx mori nucleopolyhedrovirus (BmNPV, have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143, but not BmNPV-P143 (BmP143 or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV.

  3. Characterization of A Baculovirus of Spodoptera litura (Lepidoptera : Noctuidae Isolated from Yogyakarta

    Directory of Open Access Journals (Sweden)

    Arman Wijonarko

    2007-07-01

    Full Text Available A Baculovirus has been isolated from cadaver of larvae of Spodoptera litura, a Noctuidae of agricultural pest, importance due to its wide-range hosts and the damage to their respective host. Phase contrast light microscopy observation from infected larvae showed that the fat body, hemocyte cells, and cells surrounding the trachea or tracheolus were the most tissue invaded by polyhedra. Transmission electron microscopy analysis of the occlusion body purified from diseased larva showed that the baculovirus envelope containing multiple nucleocapsid. Digestion of viral DNA with three restriction enzymes showed that the genome pattern of baculovirus isolated from Bantul were close to SpliNPV isolated from Japan and those of Spodoptera littoralis and quite distinct from those isolated from Southeast Asia region. Bioassay test performed on first to fifth instar larvae showed that the virus effectively control the young larvae, but showed some level of resistance against older larvae of Spodoptera litura.

  4. Vertical transmission of sublethal granulovirus infection in the Indian meal moth, Plodia interpunctella.

    Science.gov (United States)

    Burden, J P; Griffiths, C M; Cory, J S; Smith, P; Sait, S M

    2002-03-01

    Knowledge of the mechanisms of pathogen persistence in relation to fluctuations in host density is crucial to our understanding of disease dynamics. In the case of insect baculoviruses, which are typically transmitted horizontally via a lifestage that can persist outside the host, a key issue that remains to be elucidated is whether the virus can also be transmitted vertically as a sublethal infection. We show that RNA transcripts for the Plodia interpunctella GV granulin gene are present in a high proportion of P. interpunctella insects that survive virus challenge. Granulin is a late-expressed gene that is only transcribed after viral genome replication, its presence thus strongly indicates that viral genome replication has occurred. Almost all insects surviving the virus challenge tested positive for viral RNA in the larval and pupal stage. However, this proportion declined in the emerging adults. Granulin mRNA was also detected in both the ovaries and testes, which may represent a putative mechanism by which reduced fecundity in sublethally affected hosts might be manifested. RNA transcripts were also detected in 60-80% of second-generation larvae that were derived from mating surviving adults, but there was no difference between the sexes, with both males and females capable of transmitting a sublethal infection to their offspring. The data indicate that low-level persistent infection, with at least limited gene expression, can occur in P. interpunctella following survival of a granulovirus challenge. We believe that this is the first demonstration of a persistent, sublethal infection by a baculovirus to be initiated by a sublethal virus dose. We hypothesize that the 'latent' baculovirus infections frequently referred to in the literature may also be low level persistent, sublethal infections resulting from survival from initial baculovirus exposure.

  5. Elektron Mikroskopi dan Imunogenisitas Baculovirus oryctes Isolat Yogyakarta

    Directory of Open Access Journals (Sweden)

    YB Sumardiyono

    1995-12-01

    Full Text Available Palm rhinoceros beetle (Oryctes rhinoceros was infected per os with Yogyakarta isolate of Baculovirus oryctes in laboratory condition. Midguts of infected beetle obtained were then extracted for further nucleoprotein purification by centrifugation method. Electron microscopy studies on purified nucleoprotein revealed rod-shape viruses with rounded end measured 190×94 nm in average. One end of the particle showed tail-like structure. Antibodies against the virus were obtained by immunization to rabbit, and reacted against either purified virus or extract of infected beetle, but not against extract of healthy beetle. Key words: Baculovirus oryctes, polyclonal antibody

  6. Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

    Science.gov (United States)

    The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

  7. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Gabriel, S. E.; Stutts, M. J.

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath...

  8. Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

    International Nuclear Information System (INIS)

    Pearson, Margot N.; Rohrmann, George F.

    2004-01-01

    The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

  9. Development of serum-free media for lepidopteran insect cell lines.

    Science.gov (United States)

    Agathos, Spiros N

    2007-01-01

    Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost culture media capable of supporting good insect cell growth and ensuring high yield of baculovirus as well as the strong expression of recombinant proteins. Vertebrate sera or invertebrate hemolymph were essential supplements in first-generation insect cell media. These supplements, however, are cumbersome and expensive for routine large-scale culture; thus, their use is now circumvented by substituting the essential growth factors present in these supplements with serum-free substances. Such non-serum supplements are typically of non-animal origin and include protein hydrolysates, lipid emulsions, and specialized substances (e.g., surfactants and shear damage protecting chemicals). These supplements need to complement the defined, synthetic basal medium to ensure that the fundamental nutritional needs of the cells are satisfied. Although there is a significant number of proprietary serum-free and low-protein or protein-free media on the market, the lack of information concerning their detailed composition is a drawback in their adoption for different applications, including their adaptation to the metabolic and kinetic analysis and monitoring of a given insect cell based bioprocess. Hence, there is wide appeal for formulating serum-free media based on a rational assessment of the metabolic requirements of the lepidopteran cells during both the growth and the production phases. Techniques such as statistical experimental design and genetic algorithms adapted to the cellular behavior and the bioreactor operation mode (batch, fed-batch, or perfusion) permit the formulation of versatile serum- and protein-free media. These techniques are illustrated with recent developments of serum

  10. Propagation and Purification of Baculovirus oryctes Huger

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    1995-12-01

    Full Text Available An isolate of Baculovirus oryctes, a possible biological control agent for coconut beetle (Oryctes rhinoceros Huger from East Java was propagated and purified. The virus could be transmitted by feeding the imago with 10% sucrose containing virus from homogenate of infected beetles. Effectivity of virus to 9 healthy females by sexual copulation. Virus be succesfully purified by a method of Payne. Key words: Baculovirus oryctes, transmission, purification

  11. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    International Nuclear Information System (INIS)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-01-01

    Highlights: → All three capsid proteins can be expressed in insect cells in baculovirus expression system. → All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. → The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  12. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  13. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  14. Recombinant, catalytically inactive juvenile hormone esterase enhances efficacy of baculovirus insecticides

    NARCIS (Netherlands)

    Meer, van M.M.M.; Bonning, B.C.; Ward, V.K.; Vlak, J.M.; Hammock, B.D.

    2000-01-01

    The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses

  15. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    Science.gov (United States)

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  16. Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

    Directory of Open Access Journals (Sweden)

    Julien Thézé

    2015-04-01

    Full Text Available Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae and the baculoviruses (BVs. EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host’s immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

  17. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

    Science.gov (United States)

    Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

    2013-03-25

    Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

  18. Radioimmunoassay analysis of baculovirus granulins and polyhedrins

    International Nuclear Information System (INIS)

    Summers, M.D.; Hoops, P.

    1980-01-01

    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera. Trichoplusia ni, and Spodoptera frugiperda. Antisera were raised against Autographa californica (Ac) polyhedrin and Trichoplusia ni (Tn) granulin and analyzed for homologous and heterologous immunoreactivity by immunodiffusion and radioimmunoassay (RIA). Ac polyhedrin and Tn granulin antisera recognized antigenic determinants on several baculovirus polyhedrin and granulin proteins even though the heterologous proteins had different immunoreactivities when compared by competition radioimmunoassay. Antigenic differences among granulin and polyhedrin proteins were also detected by altered slopes of the competition reaction curves. Antiserum raised against Ac polyhedrin which was purified in the presence of SDS was tested by competition RIA for its ability to detect and react with native polyhedrin produced in the infected TN-368 cells. Ac polyhedrin antiserum had similar if not identical ability to bind to native polyhedrin and to polyhedrin purified in the presence of SDS

  19. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae, is permissive for AcMNPV infection and produces high levels of recombinant proteins

    Directory of Open Access Journals (Sweden)

    Zhang Sheng

    2010-07-01

    Full Text Available Abstract Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae. Among 8 rapidly growing lines, cell line 38 (Ao38 was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4 cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell

  20. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from

  1. Feasibility of baculovirus-mediated reporter gene delivery for efficient monitoring of islet transplantation in vivo

    International Nuclear Information System (INIS)

    Liu, Shuai; Pan, Yu; Lv, Jing; Wu, Haifei; Tian, Jingyan; Zhang, Yifan

    2014-01-01

    Objective: The objective of this study was to explore the feasibility of baculovirus vector-mediated sodium iodide symporter (NIS) gene delivery to monitor islet transplantation. Methods: Baculovirus vectors expressing green fluorescent protein (GFP) or NIS (Bac-GFP and Bac-NIS) were established using the Bac-to-Bac baculovirus expression system. The GFP expression of Bac-GFP-infected rat islets was observed in vitro by fluorescence microscopy. Iodine uptake and inhibition of iodine uptake by NaClO 4 in Bac-NIS-infected islets were dynamically monitored in vitro. Bac-GFP- or Bac-NIS-infected islets were implanted into the left axillary cavity of NOD-SCID mice, and fluorescence imaging and 125 I NanoSPECT/CT imaging were subsequently performed in vivo. Results: Bac-GFP efficiently infected rat islets (over 95% infected at MOI = 40), and the expression of GFP lasted approximately two weeks. NaClO 4 could inhibit iodine uptake by Bac-NIS-infected islets. In vivo imaging revealed that the fluorescence intensity of the transplant sites in Bac-GFP-infected groups was significantly higher than in the non-infected group. Grafts could be clearly observed by 125 I NanoSPECT/CT imaging for up to 8 h. Conclusion: Baculovirus vectors are powerful vehicles for studying rat islets in gene delivery. It is feasible to use a baculovirus vector to delivery an NIS gene for non-invasive monitoring transplanted islets in vivo by the expression of the target gene

  2. A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

    Institute of Scientific and Technical Information of China (English)

    Jian-xiaoTian; Chang-youLi; Gui-lingZheng; Guo-xunLi; PingWang; Granados

    2004-01-01

    The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.

  3. Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection

    Directory of Open Access Journals (Sweden)

    Wenqiang Wei

    2016-05-01

    Full Text Available At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

  4. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lyupina, Yulia V.; Abaturova, Svetlana B.; Erokhov, Pavel A. [N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334 (Russian Federation); Orlova, Olga V.; Beljelarskaya, Svetlana N. [V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119334 (Russian Federation); Mikhailov, Victor S., E-mail: mikhailov48@mail.ru [N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334 (Russian Federation)

    2013-02-05

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  5. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

    International Nuclear Information System (INIS)

    Lyupina, Yulia V.; Abaturova, Svetlana B.; Erokhov, Pavel A.; Orlova, Olga V.; Beljelarskaya, Svetlana N.; Mikhailov, Victor S.

    2013-01-01

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24–48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  6. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    Science.gov (United States)

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

  7. Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus

    Directory of Open Access Journals (Sweden)

    Zhu Bibo

    2012-07-01

    Full Text Available Abstract Background West Nile Virus (WNV is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells. Results In the present study, recombinant baculoviruses expressing WNV premembrane (prM and envelope (E proteins under the cytomegalovirus (CMV promoter with or without vesicular stomatitis virus glycoprotein (VSV/G were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6. Conclusions These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus.

  8. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence

  9. Analysis and functional annotation of expressed sequence tags from the fall armyworm Spodoptera frugiperda

    Science.gov (United States)

    Deng, Youping; Dong, Yinghua; Thodima, Venkata; Clem, Rollie J; Passarelli, A Lorena

    2006-01-01

    Background Little is known about the genome sequences of lepidopteran insects, although this group of insects has been studied extensively in the fields of endocrinology, development, immunity, and pathogen-host interactions. In addition, cell lines derived from Spodoptera frugiperda and other lepidopteran insects are routinely used for baculovirus foreign gene expression. This study reports the results of an expressed sequence tag (EST) sequencing project in cells from the lepidopteran insect S. frugiperda, the fall armyworm. Results We have constructed an EST database using two cDNA libraries from the S. frugiperda-derived cell line, SF-21. The database consists of 2,367 ESTs which were assembled into 244 contigs and 951 singlets for a total of 1,195 unique sequences. Conclusion S. frugiperda is an agriculturally important pest insect and genomic information will be instrumental for establishing initial transcriptional profiling and gene function studies, and for obtaining information about genes manipulated during infections by insect pathogens such as baculoviruses. PMID:17052344

  10. Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

    Science.gov (United States)

    Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

    2010-11-01

    Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  11. Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    2017-11-01

    Full Text Available Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia by targeting the cell wall components chitin and β-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

  12. Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

    Science.gov (United States)

    James McNeil; Diana Cox-Foster; James Slavicek; Kelli. Hoover

    2010-01-01

    How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its...

  13. Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno.

    Science.gov (United States)

    Rodríguez, Vanina Andrea; Belaich, Mariano Nicolás; Gómez, Diego Luis Mengual; Sciocco-Cap, Alicia; Ghiringhelli, Pablo Daniel

    2011-02-01

    Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  15. Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

    NARCIS (Netherlands)

    Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

    2006-01-01

    A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and

  16. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  17. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  18. Future vital prospect of gene expression factors of lef-7 (baculovirus expression: Old body, young cherub

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    Md. Reyad-ul-ferdous

    2018-04-01

    Full Text Available Background Baculovirus; late expression factors (Lef-7 have potential roles for protein expression in insect and mammalian cells; Efficient expression of recombinant proteins to facilitate the practical and structural investigation. Aims Lef-7 might play crucial roles in transcription and translation reactions of insect cell lines. Methods Materials and Methods: All required information regards Lef-7 was generated by exploring the internet search engine like as (PubMed, Wiley, ScienceDirect, CNKI, ACS, Google Scholar, Web of Science, SciFinder, and Baidu Scholar and libraries. Results These properties issue crucial scope for DNA cloning and act as a vital vector for insect and mammalian cells. Left-7 could be the significant site in the development of the vaccine for a couple of chronic diseases. Further investigation needs to study on therapeutic vaccines with few immunologic advantages over proteins derived from mammalian sources, and animal sources. Lef-7 demonstrates the significant impact in the fields of DNA immunology research to insight into the mechanistic and utilitarian link between autoimmunity, infectious diseases, and cancer. Conclusion This review reveals Lef-7 gene function offers a workable strategy for the expression of whole viral protomers as the future prospect of Lef-7.

  19. Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

    Science.gov (United States)

    Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

    2017-06-15

    Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases

    DEFF Research Database (Denmark)

    Petersen, Bent; Egelund, Jack; Damager, Iben

    2009-01-01

    .011 to 0.013 U (1 U = 1 nmol conversion of substrate * min(-1) * microl medium(-1)) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.......Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose to L-fucose and derivatives hereof. We have now examined expression...

  1. Baculovirus PTP2 functions as a pro-apoptotic protein

    NARCIS (Netherlands)

    Han, Yue; Houte, van Stineke; Oers, van Monique M.; Ros, Vera I.D.

    2018-01-01

    The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as

  2. Expression, purification and auto-activation of cathepsin E from insect cells.

    Science.gov (United States)

    Železnik, Tajana Z; Puizdar, Vida; Dolenc, Iztok

    2015-01-01

    Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.

  3. PLE-wu, a new member of piggyBac transposon family from insect, is active in mammalian cells.

    Science.gov (United States)

    Wu, Chunxiao; Wang, Shu

    2014-10-01

    piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  5. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

  6. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    International Nuclear Information System (INIS)

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143

  7. An immunological axis of biocontrol: infections in field-trapped insects

    Science.gov (United States)

    Tunaz, Hasan; Stanley, David

    2009-09-01

    Insect immunology is an active research arena, however, the vast majority of research in the area is conducted on model species taken from laboratory cultures. We tested the hypothesis that insects are regularly exposed to infections or invasions in nature and here report results of a field study designed to assess the extent of natural infections in insects collected from agrarian fields surrounding Kahramanmaraş, Turkey. Specimens were dissected to assess numbers of nodules. Formation of darkened, melanotic nodules is the predominant cellular immune reaction to microbial and parasitic infection, and once formed, the nodules are permanently attached to internal surfaces. The collected insects were healthy. Of the >400 examined specimens, at least some nodules were found in 98%. Numbers of nodules ranged from ˜2/individual to >100 nodules/individual. We conclude that insects are regularly challenged by microbial and parasitic infections from which they recover. The novel implication of our data is that insect immune systems may limit the host range and effectiveness of agents deployed in biological control programs. Knowledge of insect immune systems may contribute to increased use of biopesticides globally.

  8. Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

    Science.gov (United States)

    Mohan, K S; Gopinathan, K P

    1989-01-01

    Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

  9. Large scale production and downstream processing of a recombinant porcine parvovirus vaccine

    NARCIS (Netherlands)

    Maranga, L.; Rueda, P.; Antonis, A.F.G.; Vela, C.; Langeveld, J.P.M.; Casal, J.I.; Carrondo, M.J.T.

    2002-01-01

    Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of

  10. Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

    Science.gov (United States)

    Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

    2018-02-01

    Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study

    International Nuclear Information System (INIS)

    Ito, Emma; Sahri, Daniela; Knippers, Rolf; Carstens, Eric B.

    2004-01-01

    IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells

  12. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

    Science.gov (United States)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-05-27

    Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

    Science.gov (United States)

    Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

    2014-01-01

    A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  15. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

  16. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Directory of Open Access Journals (Sweden)

    Grozdea Jean J

    2002-01-01

    Full Text Available Abstract Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate, allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  17. Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

    International Nuclear Information System (INIS)

    Berretta, Marcelo F.; Deshpande, Mandar; Crouch, Erin A.; Passarelli, A. Lorena

    2006-01-01

    We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins

  18. Expression and characterization of human group C rotavirus virus-like particles in insect cells

    International Nuclear Information System (INIS)

    Clark, Kristina B.; Lin, S.-C.; Humphrey, Charles; Foytich, Kimberly; Esona, Mathew; Wang Yuhuan; Liu, Merry; Jiang Baoming

    2009-01-01

    Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.

  19. Insect biofuel cells using trehalose included in insect hemolymph leading to an insect-mountable biofuel cell.

    Science.gov (United States)

    Shoji, Kan; Akiyama, Yoshitake; Suzuki, Masato; Hoshino, Takayuki; Nakamura, Nobuhumi; Ohno, Hiroyuki; Morishima, Keisuke

    2012-12-01

    In this paper, an insect biofuel cell (BFC) using trehalose included in insect hemolymph was developed. The insect BFC is based on trehalase and glucose oxidase (GOD) reaction systems which oxidize β-glucose obtained by hydrolyzing trehalose. First, we confirmed by LC-MS that a sufficient amount of trehalose was present in the cockroach hemolymph (CHL). The maximum power density obtained using the insect BFC was 6.07 μW/cm(2). The power output was kept more than 10 % for 2.5 h by protecting the electrodes with a dialysis membrane. Furthermore, the maximum power density was increased to 10.5 μW/cm(2) by using an air diffusion cathode. Finally, we succeeded in driving a melody integrated circuit (IC) and a piezo speaker by connecting five insect BFCs in series. The results indicate that the insect BFC is a promising insect-mountable battery to power environmental monitoring micro-tools.

  20. Evolution in Oryctes baculovirus: rate and types of genomic change.

    Science.gov (United States)

    Crawford, A M; Zelazny, B

    1990-01-01

    Three cloned strains of Oryctes baculovirus were released into a previously unexposed population of the host insect, the coconut palm rhinoceros beetle, Oryctes rhinoceros. The experiment was conducted on Meemu Atoll in the Maldive Islands. Viruses were isolated from the beetle population at 1 year, 1.75 years, and 4 years after release. No changes in genotype were observed in viruses isolated after 1 and 1.75 years. After 4 years, however, three types of genomic change had occurred. A recombinant derived from two of the released strains, an isolate containing a 100-bp insert, and one example of a point mutation were found in the 22 isolates examined.

  1. Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection.

    Science.gov (United States)

    Yu, Qian; Xiong, Youhua; Gao, Hang; Liu, Jianliang; Chen, Zhiqiang; Wang, Qin; Wen, Dongling

    2015-08-04

    Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood. In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component. The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.

  2. Photorhabdus luminescens genes induced upon insect infection

    Directory of Open Access Journals (Sweden)

    Jung Kirsten

    2008-05-01

    Full Text Available Abstract Background Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. Results A differential fluorescence induction (DFI approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18 were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known

  3. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

    Directory of Open Access Journals (Sweden)

    Gale Smith

    Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

  4. New insights into HCV replication in original cells from Aedes mosquitoes.

    Science.gov (United States)

    Fallecker, Catherine; Caporossi, Alban; Rechoum, Yassine; Garzoni, Frederic; Larrat, Sylvie; François, Olivier; Fender, Pascal; Morand, Patrice; Berger, Imre; Petit, Marie-Anne; Drouet, Emmanuel

    2017-08-22

    The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

  5. How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

    Science.gov (United States)

    Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

    2010-01-20

    Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

  6. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    International Nuclear Information System (INIS)

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-01-01

    Highlights: ► Baculovirus p35 is regulated by both viral and host factors. ► Baculovirus p35 is negatively regulated by SfP53-like factor. ► Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at −1401 while P53 motif is at −1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  7. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    Energy Technology Data Exchange (ETDEWEB)

    Mohareer, Krishnaveni [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Sahdev, Sudhir [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Ranbaxy Pharmaceuticals, Gurgaon, New Delhi (India); Hasnain, Seyed E., E-mail: seh@bioschool.iitd.ac.in [Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Kusuma School of Biological Sciences, IIT Delhi, New Delhi 110016 (India); ILBS, Vasant Kunj, New Delhi (India); King Saud University, Riyadh, KSA (Saudi Arabia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  8. An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

    Science.gov (United States)

    2014-01-01

    Background Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan. Results An immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody. Conclusion A relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP. PMID:25123112

  9. Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

    Science.gov (United States)

    Shobahah, Jauharotus; Xue, Shengjie; Hu, Dongbing; Zhao, Cui; Wei, Ming; Quan, Yanping; Yu, Wei

    2017-06-19

    Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

  10. Experiment study with baculovirus-mediated transfer of the thyroid sodium/iodide symporter gene into thyroid cancer for a targeted radiotherapy

    International Nuclear Information System (INIS)

    Zhang Yifan; Li Biao; Zhao Long; You Bei; Yin Guizhi; Zhu Chengmo

    2004-01-01

    Objective: To explore the feasibility of thyroid cancers for radiotherapy by using baculoviral vector to deliver the NIS gene into the tumor cells. Method: Constructed a recombinant baculovirus encoding the human NIS gene under the control of the cytomegalovirus promoter. Using a mouse monoclonal antibody and a FITC-labeled antimouse antibody to confirm expression of the NIS protein of infected tumor cells by immunofluorescence. In vitro iodide uptake experiments were carded out on BacNIS-infected tumor cells to further characterize the BacNIS virus, and cell killing with 131I and clonogenic assay were performed on BacNIS-infected cell to observe the selective killing effect of 1311 on NIS-expressing cells. Results: Infection of thyroidcancer cells (FTC-133, W3) with BacNIS resulted in perchlorate-sensitive 125I uptake by these cells to a higher level than that in noninfected cells. But 1251 uptake of 8505C is very low. Demonstrating that the BacNIS vector can function in tumor cells. In addition, AdNIS-infected tumor cells were selectively killed by exposure to 1311, as revealed by clonogenicassays, higher than that in nontreated tumors. Conclusions: AdNIS is very efficient in triggering iodide uptake by infected tumor cell, outlining the potential of this novel cancer gene therapy approach for a targeted radiotherapy. (authors)

  11. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

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    Aurélien Chateigner

    2015-07-01

    Full Text Available Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%. K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs. Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  12. Analysis of whitefly transcriptional responses to Beauveria bassiana infection reveals new insights into insect-fungus interactions.

    Science.gov (United States)

    Xia, Jun; Zhang, Chang-Rong; Zhang, Shan; Li, Fang-Fang; Feng, Ming-Guang; Wang, Xiao-Wei; Liu, Shu-Sheng

    2013-01-01

    The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis. Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection. To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly

  13. Baculovirus p35 increases pancreatic β-cell resistance to apoptosis

    International Nuclear Information System (INIS)

    Hollander, Kenneth; Bar-Chen, Michal; Efrat, Shimon

    2005-01-01

    β-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in β-cells exposed to cytokines and hIAPP, as well as the effects of p35 on β-cell function. The p35 gene was introduced into βTC-tet cells, a differentiated murine β-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted β-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from β-cell damage caused by hIAPP in type 2 diabetes

  14. Response to Dengue virus infections altered by cytokine-like substances from mosquito cell cultures

    Directory of Open Access Journals (Sweden)

    Laosutthipong Chaowanee

    2010-11-01

    Full Text Available Abstract Background With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells. Results Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2 and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects. Conclusions This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.

  15. Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda.

    Science.gov (United States)

    Barrera, Gloria Patricia; Belaich, Mariano Nicolás; Patarroyo, Manuel Alfonso; Villamizar, Laura Fernanda; Ghiringhelli, Pablo Daniel

    2015-11-25

    Baculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use. Genome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus' life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport. The SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring

  16. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    International Nuclear Information System (INIS)

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-01-01

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  17. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

    Science.gov (United States)

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

    2016-01-15

    Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an

  18. Initiative to manufacture and characterize Baculovirus Reference Material

    NARCIS (Netherlands)

    Kamen, A.; Aucoin, M.; Merten, O.W.; Alves, P.C.M.; Hashimoto, Y.; Airenne, K.; Hu, Y.C.; Mezzina, M.; Oers, van M.M.

    2011-01-01

    This letter to the editor brings to the attention of researchers an initiative to develop a baculovirus reference material repository. To be successful this initiative needs the support of a broad panel of researchers working with baculovirus vectors for recombinant protein production and gene

  19. Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

    Science.gov (United States)

    Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

    2004-10-01

    Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

  20. Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

    Science.gov (United States)

    Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

    Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

  1. Pemanfaatan Antibodi Monoklonal dalam Immunoassay untuk Deteksi Baculovirus oryctes

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    2000-12-01

    Full Text Available The application of non-precoated Indirect Enzyme-Linked Immunosorbent Assay (ELISA and Dot Immunobinding Assay (DIBA employing monoclonal antibodies (MCA against Yogyakarta isolate of Baculovirus oryctes Huger. was described. The MCA-Bv-4 having subclass of IgG2a and titer in vitro of 10^4 - 10^5 proved to be useful antibody for virus detection. The great potential of I-ELISA using MCA-Bv-4 has been it's specificity being able to discriminate between healthy an virus-infected coconut beetle (Oryctes rhinoceros L.. The assay could also differentiate between B. oryctes and Monodon baculovirus, the pathogen of shrimp disease. The best tissue for preparing virus antigen from crude extract was found to be the midgut of the beetle. Head, thorax, abdomen and tibia were not suitable for preparing the test antigen. The crude extract of beetle showed high endogenous enzyme activity to the substrate of DIBA, which precluded the detection of B. oryctes using DIBA. The MCA-Bv-4 could be used to improve the monitoring of the virus to support the program of biological control of coconut beetle using B. oryctes.

  2. Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

    DEFF Research Database (Denmark)

    Victor, Michala E; Bengtsson, Anja; Andersen, Gorm

    2010-01-01

    -exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w...... PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed....

  3. Wolbachia infection complexity among insects in the tropical rice-field community.

    Science.gov (United States)

    Kittayapong, P; Jamnongluk, W; Thipaksorn, A; Milne, J R; Sindhusake, C

    2003-04-01

    Wolbachia are a group of intracellular bacteria that cause reproductive alterations in their arthropod hosts. Widely discordant host and Wolbachia phylogenies indicate that horizontal transmission of these bacteria among species sometimes occurs. A likely means of horizontal transfer is through the feeding relations of organisms within communities. Feeding interactions among insects within the rice-field insect community have been well documented in the past. Here, we present the results of a polymerase chain reaction-based survey and phylogenetic analysis of Wolbachia strains in the rice-field insect community of Thailand. Our field survey indicated that 49 of 209 (23.4%) rice-field insect species were infected with Wolbachia. Of the 49 infected species, 27 were members of two feeding complexes: (i) a group of 13 hoppers preyed on by 2 mirid species and parasitized by a fly species, and (ii) 2 lepidopteran pests parasitized by 9 wasp species. Wolbachia strains found in three hoppers, Recilia dorsalis, Nephotettix malayanus and Nisia nervosa, the two mirid predators, Cyrtorhinus lividipennis and Tytthus chinensis, and the fly parasitoid, Tomosvaryella subvirescens, were all in the same Wolbachia clade. In the second complex, the two lepidopteran pests, Cnaphalocrocis medinalis and Scirpophaga incertulas, were both infected with Wolbachia from the same clade, as was the parasitoid Tropobracon schoenobii. However, none of the other infected parasitoid species in this feeding complex was infected by Wolbachia from this clade. Mean (+/- SD) genetic distance of Wolbachia wsp sequences among interacting species pairs of the hopper feeding complex (0.118 +/- 0.091 nucleotide sequence differences), but not for the other two complexes, was significantly smaller than that between noninteracting species pairs (0.162 +/- 0.079 nucleotide sequence differences). Our results suggest that some feeding complexes, such as the hopper complex described here, could be an important

  4. 寨卡病毒E蛋白在重组杆状病毒中的表达%Expression of envelope protein of Zika virus in baculovirus expression system

    Institute of Scientific and Technical Information of China (English)

    高寒春; 姚立红; 王超; 郑丽舒

    2017-01-01

    目的 在杆状病毒中表达寨卡病毒(Zika virus,ZIKV)包膜蛋白(Envelope protein,E蛋白).方法 人工合成ZIKV E基因全长序列(1 518 bp),并克隆到pFastBac1载体上,得到重组杆状病毒转移载体pFB1-E,转化含DH10Bac感受态细胞,获得骨架质粒rBacmid-E,转染sf9细胞,得到重组杆状病毒rBac-E.检测病毒滴度,PCR法检测E基因的插入,间接免疫荧光和Western blot法检测E蛋白的表达.结果 经PCR方法鉴定,重组杆状病毒骨架质粒构建成功,重组病毒rBac-E(第3代)病毒滴度为2.58×105pfu/ml.提取感染rBac-E的sf9细胞基因组,经PCR扩增得到3 830 bp的条带,间接免疫荧光检测出现特异性绿色荧光.Western blot鉴定可见感染重组杆状病毒rBac-E的细胞沉淀中在相对分子质量55×103处有特异性条带.结论 在杆状病毒表达系统中重组表达了ZIKV E蛋白,为ZIKV E蛋白功能研究及疫苗的开发奠定了基础.%Objective To express envelope protein of ZIKA virus in baculovirus expression system.Methods Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1.The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells.The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells,and the constructed recombinant baculovirus rBac-E was determined for titer,for insertion of E gene by PCR,and for expression of E protein by IFA and Western blotting.Results PCR proved that skeleton plasmid rBacmid-E was constructed correctly.The titer of rBac-E of passage 3 was 2.58 × 105 pfu/ ml.The genome of infected cells virus was extracted,the gene band at length of 3 830 bp was observed after PCR amplification.Indirect immunofluorescence of the infected cells showed the specific green fluorescence,55 × 103 specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBacE.Conclusions The recombinant

  5. Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy.

    Science.gov (United States)

    Negrete, Alejandro; Esteban, Geoffrey; Kotin, Robert M

    2007-09-01

    A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 x 10(15) particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 x 10(16) particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

  6. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

    Science.gov (United States)

    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Increasing cell-device adherence using cultured insect cells for receptor-based biosensors

    Science.gov (United States)

    Terutsuki, Daigo; Mitsuno, Hidefumi; Sakurai, Takeshi; Okamoto, Yuki; Tixier-Mita, Agnès; Toshiyoshi, Hiroshi; Mita, Yoshio; Kanzaki, Ryohei

    2018-03-01

    Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell-device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell-device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell-device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs.

  8. The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

    Science.gov (United States)

    Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

    2016-06-01

    A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

  9. A novel approach to biocontrol: Release of live insect hosts pre-infected with entomopathogenic nematodes.

    Science.gov (United States)

    Gumus, Arife; Karagoz, Mehmet; Shapiro-Ilan, David; Hazir, Selcuk

    2015-09-01

    As a new application approach, we tested the efficacy of releasing live insect hosts that were pre-infected with entomopathogenic nematodes against insect pests living in cryptic habitats. We hypothesized that the pre-infected hosts could carry the next generation of emerging nematode infective juveniles to hard-to-reach target sites, and thereby facilitate enhanced control in cryptic habitats. Thus, the infected hosts act as "living insect bombs" against the target pest. We tested this approach using two model insect pests: a chestnut tree pest, the goat moth Cossus cossus (Lepidiptera: Cossidae), and a lawn caterpillar, Spodoptera cilium (Lepidoptera: Noctuidae). One pest is considered hard-to-reach via aqueous spray (C. cossus) and the other is more openly exposed in the environment (S. cilium). C. cossus and S. cilium studies were conducted in chestnut logs and Bermudagrass arenas, respectively. The living bomb approach was compared with standard nematode application in aqueous spray and controls (without nematode application); Steinernema carpocapsae (Rize isolate) was used in all experiments. The percentage larval mortality of C. cossus was 86% in the living insect bomb treatment, whereas, all other treatments and controls exhibited less than 4% mortality. The new approach (living bomb) was equally successful as standard aqueous application for the control of S. cilium larvae. Both methods exhibited more than 90% mortality in the turfgrass arena. Our new approach showed an immense potential to control insect pests living in hard-to-reach cryptic habitats. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2008-02-01

    Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

  11. Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

    Science.gov (United States)

    Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

    2015-01-01

    The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

  12. Baculovirus-expressed constructs induce immunoglobulin G that recognizes VAR2CSA on Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Barfod, Lea; Nielsen, Morten A; Turner, Louise

    2006-01-01

    We raised specific antisera against recombinant VAR2CSA domains produced in Escherichia coli and in insect cells. All were reactive in enzyme-linked immunosorbent assay, but only insect cell-derived constructs induced immunoglobulin G (IgG) that was reactive with native VAR2CSA on the surface...

  13. Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

    DEFF Research Database (Denmark)

    Sørensen, K.J.; de Stricker, K.; Dyrting, K.C.

    2005-01-01

    A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector...... infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge...... with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs...

  14. Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV).

    Science.gov (United States)

    Sudhakaran, R; Haribabu, P; Kumar, S Rajesh; Sarathi, M; Ahmed, V P Ishaq; Babu, V Sarath; Venkatesan, C; Hameedl, A S Sahul

    2008-04-01

    Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for MrNV and XSV transmission to M. rosenbergii.

  15. Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

    Directory of Open Access Journals (Sweden)

    Junmin Li

    Full Text Available Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi which generates viral-derived small interfering RNAs (siRNAs. However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus was infected by Rice black-streaked dwarf virus (RBSDV (Reoviridae; Fijivirus, more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV, a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

  16. Baculovirus display of single chain antibody (scFv using a novel signal peptide

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  17. Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

    Science.gov (United States)

    McGonigle, John E; Purves, Joanne; Rolff, Jens

    2016-06-01

    Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The Drosophila gene brainiac encodes a glycosyltransferase putatively involved in glycosphingolipid synthesis

    DEFF Research Database (Denmark)

    Schwientek, Tilo; Keck, Birgit; Levery, Steven B

    2002-01-01

    -linked mannose as well as beta-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galbeta1-4Glcbeta1-Cer) and insects (Manbeta1-4Glcbeta1-Cer) are different. Both disaccharide glycolipids served as substrates for brainiac, but glycolipids of insect cells...... have so far only been found to be based on the GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer core structure. Infection of High Five(TM) cells with baculovirus containing full coding brainiac cDNA markedly increased the ratio of GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer glycolipids compared with Galbeta1-4Manbeta1......-4Glcbeta1-Cer found in wild type cells. We suggest that brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids....

  19. Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

    Science.gov (United States)

    Yang, Z; Hu, Y; Yuan, P; Yang, Y; Wang, K; Xie, L Y; Huang, S L; Liu, J; Ran, L; Song, Z H

    2018-03-01

    In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice. Copyright© by the Polish Academy of Sciences.

  20. Granulosis viruses, with emphasis on the GV of the Indian meal moth, Plodia interpunctella.

    Science.gov (United States)

    Consigli, R A; Tweeten, K A; Anderson, D K; Bulla, L A

    1983-01-01

    The granulosis viruses and nuclear polyhedrosis viruses are being considered for use as biological insecticides for control of their insect hosts. Many of these insect species, which include some of the most serious pests of agriculture and forests, have become difficult to control because they have developed resistance to chemical insecticides. Several laboratory and field studies have demonstrated that the baculoviruses (GV and NPV) are promising alternatives to chemicals for the control of economically important insects. These viruses are highly virulent, selective, and stable, and the impact on the environment following their application is minimal. A decision concerning the application of baculoviruses to stored grain and field crops must be based upon a prudent consideration of the benefits to be obtained and the potential risks of their use. Such decisions should be made only after consideration of the physical, chemical, and biological properties of these viruses. In addition, methods must be developed for the unequivocal identification of these viruses, and their effects on nontarget species at the cellular and molecular levels must be investigated. This can best be accomplished if a sufficient body of knowledge regarding the molecular properties of these viruses and their infection process is accumulated by an extensive quantitative approach. Much of this knowledge is lacking because, prior to their consideration for use as insecticides, the baculoviruses appeared to have little medical or economic importance. As a result, interest in studying them was limited. It has become obvious that the molecular properties of these viruses must be investigated if full advantage is to be taken of using them as insect control agents, and if present and future problems concerning their use as insecticides are to be handled properly. Fundamental research on the biochemical and biophysical properties of baculoviruses has concentrated mainly on a variety of nuclear

  1. Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink

    DEFF Research Database (Denmark)

    Christensen, J; Alexandersen, Søren; Bloch, B.

    1994-01-01

    The VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2...... protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly...

  2. Characterization of the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome : a (phylo)genetic study

    NARCIS (Netherlands)

    Hu, Z.

    1998-01-01

    Baculoviruses are attractive biological alternatives to chemical insecticides for insect pest control. So far, more than 600 baculoviruses have been isolated from different insect species which provide a rich resource for developing new viral insecticides. Most baculoviruses are host

  3. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    Science.gov (United States)

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

  4. Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda).

    Science.gov (United States)

    Martins, E S; Aguiar, R W D S; Martins, N F; Melatti, V M; Falcão, R; Gomes, A C M M; Ribeiro, B M; Monnerat, R G

    2008-05-01

    To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.

  5. Neutralizing antibodies induced by recombinant virus-like particles of enterovirus 71 genotype C4 inhibit infection at pre- and post-attachment steps.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Ku

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. METHODS/FINDINGS: In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs. Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. CONCLUSIONS: Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

  6. Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication.

    Science.gov (United States)

    Asser-Kaiser, Sabine; Radtke, Pit; El-Salamouny, Said; Winstanley, Doreen; Jehle, Johannes A

    2011-02-20

    An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGV(hsp-eGFP)) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. The entomopathogenic fungus Metarhizium robertsii communicates with the insect host Galleria mellonella during infection.

    Science.gov (United States)

    Mukherjee, Krishnendu; Vilcinskas, Andreas

    2018-01-01

    Parasitic fungi are the only pathogens that can infect insect hosts directly through their proteinaceous exoskeleton. Penetration of the cuticle requires the release of fungal enzymes, including proteinases, which act as virulence factors. Insects can sense fungal infections and activate innate immune responses, including the synthesis of antifungal peptides and proteinase inhibitors that neutralize the incoming proteinases. This well-studied host response is epigenetically regulated by histone acetylation/deacetylation. Here we show that entomopathogenic fungi can in turn sense the presence of insect-derived antifungal peptides and proteinase inhibitors, and respond by inducing the synthesis of chymotrypsin-like proteinases and metalloproteinases that degrade the host-derived defense molecules. The rapidity of this response is dependent on the virulence of the fungal strain. We confirmed the specificity of the pathogen response to host-derived defense molecules by LC/MS and RT-PCR analysis, and correlated this process with the epigenetic regulation of histone acetylation/deacetylation. This cascade of responses reveals that the coevolution of pathogens and hosts can involve a complex series of attacks and counterattacks based on communication between the invading fungal pathogen and its insect host. The resolution of this process determines whether or not pathogenesis is successful.

  8. Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals.

    Science.gov (United States)

    Dalla, Safaa; Swarts, Herman G P; Koenderink, Jan B; Dobler, Susanne

    2013-12-01

    Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    Science.gov (United States)

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  10. Plant Virus–Insect Vector Interactions: Current and Potential Future Research Directions

    Science.gov (United States)

    Dietzgen, Ralf G.; Mann, Krin S.; Johnson, Karyn N.

    2016-01-01

    Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors. PMID:27834855

  11. Autographa californica Multicapsid Nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH

    NARCIS (Netherlands)

    Dong, S.; Wang, M.; Qiu, Z.; Deng, F.; Vlak, J.M.; Hu, Z.H.; Wang, H.L.

    2010-01-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue

  12. Validation of Aedes aegypti Aag-2 cells as a model for insect immune studies.

    Science.gov (United States)

    Barletta, Ana Beatriz Ferreira; Silva, Maria Clara L Nascimento; Sorgine, Marcos H Ferreira

    2012-07-24

    The understanding of mosquito immune responses can provide valuable tools for development of novel mosquito control strategies. Aiming the study at insect innate immunity, continuous insect cell lines have been established and used as research tools due to the fact that they constitute more homogeneous, sensitive, and reproducible systems than the insects from which they originated. More recently, Aag-2, an Aedes aegypti cell lineage, began to be frequently used as a model for studies of mosquito immunity. Nevertheless, to our knowledge, no study has systematically characterized the responses of Aag-2 cell line against different kinds of pathogens and compared its response to those exhibited by whole mosquitoes. For this reason, in this study we characterized gene expression profiles of the Aag-2 cell line in response to different kinds of immune challenges, such as Gram negative and positive bacteria, fungi and viruses, comparing the obtained results with the ones already described in the literature for whole mosquitoes. Aedes aegypti Aag-2 cells were exposed to different immune stimuli (gram-positive and gram negative heat inactivated bacteria, zymosan or Sindbis virus) for 24 hours and the expression of selected marker genes from toll, IMD and Jak/STAT pathways was analyzed by qPCR. Also, cells were incubated with fluorescent latex beads for evaluation of its phagocytosis capacity. Aag-2 cells were stimulated with two concentrations of heat-killed Gram negative (Enterobacter cloacae) or Gram positive (Micrococcus luteus) bacteria, Zymosan or infected with Sindbis virus and the expression of key genes from the main immune related pathways, Toll, IMD and Jak/STAT, were investigated. Our results suggest that Toll and IMD pathways are activated in response to both Gram positive and negative bacteria and Zymosan in Aag-2 cells, displaying an immune profile similar to those described in the literature for whole mosquitoes. The same stimuli were also capable of

  13. Antimicrobial defence and persistent infection in insects revisited.

    Science.gov (United States)

    Makarova, Olga; Rodríguez-Rojas, Alexandro; Eravci, Murat; Weise, Chris; Dobson, Adam; Johnston, Paul; Rolff, Jens

    2016-05-26

    Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitroThis article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'. © 2016 The Author(s).

  14. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  15. Insect immunology and hematopoiesis.

    Science.gov (United States)

    Hillyer, Julián F

    2016-05-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and activate effector pathways. Among the immune signaling pathways are the Toll, Imd, Jak/Stat, JNK, and insulin pathways. Activation of these and other pathways leads to pathogen killing via phagocytosis, melanization, cellular encapsulation, nodulation, lysis, RNAi-mediated virus destruction, autophagy and apoptosis. This review details these and other aspects of immunity in insects, and discusses how the immune and circulatory systems have co-adapted to combat infection, how hemocyte replication and differentiation takes place (hematopoiesis), how an infection prepares an insect for a subsequent infection (immune priming), how environmental factors such as temperature and the age of the insect impact the immune response, and how social immunity protects entire groups. Finally, this review highlights some underexplored areas in the field of insect immunobiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

    Science.gov (United States)

    Wang, Meng; Zheng, Zhenhua; Meng, Jin; Wang, Han; He, Man; Zhang, Fuxian; Liu, Yan; Hu, Bin; He, Zike; Hu, Qinxue; Wang, Hanzhong

    2015-09-01

    Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 μg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

    Science.gov (United States)

    Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

    2014-01-01

    Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

  18. Laboratory trials to infect insects and nematodes by some acaropathogenic Hirsutella strains (Mycota: Clavicipitaceous anamorphs).

    Science.gov (United States)

    Bałazy, Stanisław; Wrzosek, Marta; Sosnowska, Danuta; Tkaczuk, Cezary; Muszewska, Anna

    2008-02-01

    Laboratory assays have been carried out to artificially infect insect larvae of the birch bark-beetle (Scolytus ratzeburgi Jans.-Coleoptera, Scolytidae) and codling moth Cydia pomonella L. -Lepidoptera, Tortricidae) as well as the potato cyst nematode-Globodera rostochiensis Wollenweber, sugar beet nematode-Heterodera schachtii Schmidt and root-knot nematode-Meloidogyne hapla Chif (Nematoda, Heteroderidae), by the phialoconidia of some fungal species of the genus Hirsutella. From among four species tested on insects only H. nodulosa Petch infected about 20% of S. ratzeburgi larvae, whereas H. kirchneri (Rostrup) Minter, Brady et Hall, H. minnesotensis Chen, Liu et Chen, and H. rostrata Bałazy et Wiśniewski did not affect insect larvae. Only single eggs of the root-knot nematode were infected by H. minnesotensis in the laboratory trials, whereas its larvae remained unaffected. No infection cases of the potato cyst nematode (G. rostochiensis) and sugar beet nematode eggs were obtained. Comparisons of DNA-ITS-region sequences of the investigated strains with GenBank data showed no differences between H. minnesotensis isolates from the nematodes Heterodera glycines Ichinohe and from tarsonemid mites (authors' isolate). A fragment of ITS 2 with the sequence characteristic only for H. minnesotensis was selected. Two cluster analyses indicated close similarity of this species to H. thompsonii as sister clades, but the latter appeared more heterogenous. Insect and mite pathogenic species H. nodulosa localizes close to specialized aphid pathogen H. aphidis, whereas the phytophagous mite pathogens H. kirchneri and H. gregis form a separate sister clade. Hirsutella rostrata does not show remarkable relations to the establishment of aforementioned groups. Interrelated considerations on the morphology, biology and DNA sequencing of investigated Hirsutella species state their identification more precisely and facilitate the establishment of systematic positions.

  19. Functional analysis of the baculovirus 10 kilodalton protein

    NARCIS (Netherlands)

    Oers, van M.M.

    1994-01-01

    Nuclear polyhedrosis viruses belong to the family Baculoviridae and cause fatal diseases in arthropods predominantly in insects of the order Lepidoptera . These viruses were first reported in silk worms where viral infections could have devastating

  20. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    International Nuclear Information System (INIS)

    Nordlund, Henri R.; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

    2005-01-01

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches

  1. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

    Science.gov (United States)

    Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

    2005-10-14

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

  2. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    Science.gov (United States)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  3. Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

    DEFF Research Database (Denmark)

    Madsen, Eva Smedegaard; Madsen, Knud Gert; Nielsen, Jens

    1997-01-01

    The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immune sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2...... was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2...... producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies...

  4. Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Yokoo, Masako [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Fujita, Ryosuke [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021 (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213 (Japan); Yoshimizu, Mamoru; Kasai, Hisae [Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611 (Japan); Asano, Shin-ichiro [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Bando, Hisanori, E-mail: hban@abs.agr.hokudai.ac.jp [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan)

    2013-09-13

    Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

  5. Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

    International Nuclear Information System (INIS)

    Yokoo, Masako; Fujita, Ryosuke; Nakajima, Yumiko; Yoshimizu, Mamoru; Kasai, Hisae; Asano, Shin-ichiro; Bando, Hisanori

    2013-01-01

    Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells

  6. Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

    Science.gov (United States)

    Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

    2015-12-02

    Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Radiosensitivity of cultured insect cells: I. Lepidoptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D 0 , d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D 0 of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects

  8. Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System

    KAUST Repository

    Morokuma, Daisuke

    2017-03-24

    Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.

  9. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    Science.gov (United States)

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  10. [Anti-infective defence strategies and methods of escape from entomologic pathogens under immunologic control of insects].

    Science.gov (United States)

    Jarosz, J

    1996-01-01

    Insect immunity comprises a complex of several distinct systems, both haemocytic and humoral in nature, that cooperate together in a more or less coordinated way to provide protection of the body cavity from invading microorganisms. Insects can respond to infections by a selective synthesis of haemolymph immune proteins that are responsible for antibacterial immunity. Antibacterial activity of insect blood is attributable to innate compounds such as lysozome, and to induced polypeptides or small basic proteins absent in non-immunized insects. The cecropins and attacins in Lepidoptera, and diptericins in Diptera are the inducible antibacterial immune proteins well defined biochemically. Bacterial pathogens and some parasites of insects, preferably entomogenous rhabditid nematodes, have developed the mechanism by which they may counteract insect immunity. This phenomenon is realized either by escaping immune reactions or by degrading antimicrobial factors of haemolymph in an active process. Passive resistance of parasites to insect immunity is a result of a strong evolutionary pressure on parasites to develop mechanisms to escape insect immune reactions or to minimize their effectiveness through changes in the parasite itself. Active resistance to the insect non-self response system involves a partial or total destruction of immune proteins by extracellular proteinases released during parasitism.

  11. Advances in Synthetic Peptides Reagent Discovery

    Science.gov (United States)

    2013-07-01

    phage [4], bacteria [5-7], yeast [8, 9], insect cell/baculovirus [10], as well as mammalian cell culture [11]. Although each system has its unique...dependent characteristics,” The Journal of Immunology , 147(10), 3545-3552 (1991). [32] S. Becker, H. Höbenreich, A. Vogel et al., “Single‐Cell High

  12. Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

    Science.gov (United States)

    The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

  13. Horizontal Transmission of Intracellular Insect Symbionts via Plants

    Directory of Open Access Journals (Sweden)

    Ewa Chrostek

    2017-11-01

    Full Text Available Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

  14. Baculovirus potential for agricultural pests management in Cuba

    Directory of Open Access Journals (Sweden)

    Jorge Luis Ayala Sifontes

    2017-10-01

    Full Text Available Cuba has an international reputation for implementing widespread biological control of pests, and microbial biocontrol is an integral component of most pest management programs. One class of microbial pesticides however, has not been developed in Cuba, bio-insecticides based on the Baculoviridae. This class of safe and environmentally protective microbial pesticides is used ever more commonly worldwide as an alternative to chemical pesticides. The characteristics of the viruses of this family, particularly their high host specificity, safety to non-target organisms, capacity to persist in nature and create epizootics, and the economy with which they can be produced "in vivo", all make them attractive for incorporation into pest management programs along with other pesticides developed in Cuba. The mass production technology is well understood in Cuba and biofactories already exist for a number of microbial biocontrol products. In the province of Sancti Spíritus, the Plant Protection Laboratory of the Ministry of Agriculture, with the cooperation of the Institute for Sustainable Horticulture, Kwantlen Polytechnic University, are resuming the work which began in the 90´s to develop baculovirus products in support of sustainable agriculture in Cuba. This work is being carried out with the participation of young Canadian and Cuban students and professionals. The program includes research with the multicapsid nuclear polyhedrosis viruses of Spodoptera frugiperda (SfMNPV and S. exigua (SeMNPV and the search for native isolates of Baculovirus in Plutella xylostella, three priority pests in Cuba. In other jurisdictions they are well controlled by baculoviruses, and the expectation is that this same result is possible in Cuba.

  15. Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

    NARCIS (Netherlands)

    Long, G.; Pan, M.; Westenberg, M.; Vlak, J.M.

    2006-01-01

    F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD),

  16. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

    DEFF Research Database (Denmark)

    Nielsen, Morten A; dos Santos Marques Resende, Mafalda; de Jongh, Willem A

    2015-01-01

    -functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant...... with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found...

  17. Comparative venom toxicity between Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) toward the hemocytes of their natural hosts, non-target insects and cultured insect cells.

    Science.gov (United States)

    Zhang, Zhong; Ye, Gong-Yin; Cai, Jun; Hu, Cui

    2005-09-01

    Crude venoms from two parasitoid species, Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) were assayed for biological activities toward hemocytes from two species of their natural hosts and eight species of their non-natural hosts as well as two lines of cultured Lepidoptera cells, respectively. By inhibiting the spreading and viability of insect hemocytes, the venom from P. puparum displayed significantly higher activities toward plasmatocytes and granular cells from both larvae and pupae of two natural hosts, Pieris rapae and Papilio xuthus, and lower activity toward those from Spodoptera litura, Musca domestica and Sarcophaga peregrina. However, no effect was found towards any type of hemocytes from other five insects tested, namely, Ectropis oblique, Galleria mellonella, Sesamia inferens, Bombyx mori and Parnara guttata. In contrast, the venom from N. vitripennis showed a narrower range of targeted insects. It appeared to have highly adverse effects on the spreading and viability of plasmatocytes and granular cells only from the natural hosts, M. domestica and S. peregrina, little toxicity to cells from P. rapae and P. xuthus, and no effect on any of the other insects tested. Pteromalus puparum venom also apparently presented a high ability to block the spreading of Tn-5B1-4 cells derived from Trichoplusia ni, and high cytotoxicity to the cells and Ha cells derived from Helicoverpa armigera. Nasonia vitripennis venom, however, only had a marked lethal effect to Ha cells. In addition, the possibility that the host range of a defined parasitoid could be assessed using our method of treating hemocytes from candidate insects with venom in vitro, and the potential of our venoms tested in the development of bio-insecticides, insect-resistant transgenic plants, are discussed.

  18. Towards a molecular identification and classification system of lepidopteran-specific baculoviruses

    International Nuclear Information System (INIS)

    Lange, Martin; Wang Hualin; Hu Zhihong; Jehle, Johannes A.

    2004-01-01

    Virus genomics provides novel approaches for virus identification and classification. Based on the comparative analyses of sequenced lepidopteran-specific baculovirus genomes, degenerate oligonucleotides were developed that allow the specific amplification of several regions of the genome using polymerase chain reaction (PCR) followed by DNA sequencing. The DNA sequences within the coding regions of three highly conserved genes, namely polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8), and late expression factor 9 (lef-9), were targeted for amplification. The oligonucleotides were tested on viral DNAs isolated from historical field samples, and amplification products were generated from 12 isolated nucleopolyhedrovirus (NPV) and 8 granulovirus (GV) DNAs. The PCR products were cloned or directly sequenced, and phylogenetic trees were inferred from individual and combined data sets of these three genes and compared to a phylogeny, which includes 22 baculoviruses using a combined data set of 30 core genes. This method allows a fast and reliable detection and identification of lepidopteran-specific NPVs and GVs. Furthermore, a strong correlation of the base composition of these three genome areas with that of the complete virus genome was observed and used to predict the base composition of uncharacterized baculovirus genomes. These analyses suggested that GVs have a significantly higher AT content than NPVs

  19. Insects vis a vis radiations

    International Nuclear Information System (INIS)

    Srivastava, Meera

    2014-01-01

    Insects have turned out to be much more radiation resistant. For most insects a dose of about 500-700 Gy is required to kill them within a few weeks of exposure; although cockroaches require 900-1000 Gy. Killing insects in less than a few days requires much higher doses. These doses are for mature insects, the immature stages of some insects can be killed by doses as low as 40 Gy. Some insects can be sterilized at even lower doses, and this has application in insect control. Screw-worms, for example, can be sterilized with doses of 25-50 Gy. By contrast, doses as low as 3 Gy caused death of humans in Hiroshima and Nagasaki and doses of about 6 Gy caused death of fire fighters in the Chernobyl accident. It is not exactly certain what the basis is for the resistance of insects to ionizing radiation. It is not animal size by itself, nor lack of penetration. It is also not because of few dividing cells as these are more radiosensitive than non-dividing ones. The speculation that insects might have lower oxygen tensions, and the lack of oxygen is known to protect cells from radiation also does not work. Insect cells might have an enhanced capacity to repair radiation damage also could not be proven. The number of chromosomes influenced radio-sensitivity, and that insects had fewer chromosomes could be true. The radiation resistance is inherent to the cells, since cells derived from insects are also radiation resistant when grown in cell culture. For example, a dose of 60 Gy is required to produce a 80% kill of insect cells, while doses of 1-2 Gy are sufficient to generate this level of killing in mammalian cells. But, nevertheless, according to recent researches, radiation from Japan's leaking Fukushima nuclear plant has caused mutations in some butterflies. It is therefore clear that insects are resistant to ionizing radiation and that this resistance is an inherent property of their cells. But it is not clear exactly what the basis of this cellular resistance is

  20. Biology and genomics of viruses within the genus Gammabaculovirus.

    Science.gov (United States)

    Arif, Basil; Escasa, Shannon; Pavlik, Lillian

    2011-11-01

    Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

  1. Perturbed cholesterol and vesicular trafficking associated with dengue blocking in Wolbachia-infected Aedes aegypti cells.

    Science.gov (United States)

    Geoghegan, Vincent; Stainton, Kirsty; Rainey, Stephanie M; Ant, Thomas H; Dowle, Adam A; Larson, Tony; Hester, Svenja; Charles, Philip D; Thomas, Benjamin; Sinkins, Steven P

    2017-09-13

    Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders. Wolbachia infection of mosquitoes can block dengue virus infection and is tested in field trials, but the mechanism of action is unclear. Using proteomics, Geoghegan et al. here identify effects of Wolbachia on cholesterol homeostasis and dengue virus replication in Aedes aegypti.

  2. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Science.gov (United States)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  3. Chimaeric Virus-Like Particles Derived from Consensus Genome Sequences of Human Rotavirus Strains Co-Circulating in Africa

    Science.gov (United States)

    Jere, Khuzwayo C.; O'Neill, Hester G.; Potgieter, A. Christiaan; van Dijk, Alberdina A.

    2014-01-01

    Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented

  4. Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

    Science.gov (United States)

    Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

    2015-09-14

    We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

    Science.gov (United States)

    Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

    1998-03-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

  6. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, Toshiki [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Shibayama, Naoya [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Yoon, Young-Ho [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Yun, Kyung-Mook [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Hamamoto, Toshiro [Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Tame, Jeremy R. H.; Park, Sam-Yong, E-mail: park@tsurumi.yokohama-cu.ac.jp [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan)

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  7. Pathogen avoidance by insect predators

    OpenAIRE

    Meyling, Nicolai V.; Ormond, Emma; Roy, Helen E.; Pell, Judith K.

    2008-01-01

    Insects can detect cues related to the risk of attack by their natural enemies. Pathogens are among the natural enemies of insects and entomopathogenic fungi attack a wide array of host species. Evidence documents that social insects in particular have adapted behavioural mechanisms to avoid infection by fungal pathogens. These mechanisms are referred to as 'behavioural resistance'. However, there is little evidence for similar adaptations in non-social insects. We have conducted experime...

  8. Vaccination of carp against SVCV with an oral DNA vaccine or an insect cells-based subunit vaccine.

    Science.gov (United States)

    Embregts, C W E; Rigaudeau, D; Tacchi, L; Pijlman, G P; Kampers, L; Veselý, T; Pokorová, D; Boudinot, P; Wiegertjes, G F; Forlenza, M

    2018-03-19

    We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Radiosensitivity of cultured insect cells: II. Diptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D 0 values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells

  10. Infection of Drosophila suzukii with the obligate insect-pathogenic fungus Entomophthora muscae

    DEFF Research Database (Denmark)

    Becher, Paul G.; Jensen, Rasmus E.; Natsopoulou, Myrsini E.

    2018-01-01

    Physiological constraints restrict specialist pathogens from infecting new hosts. From an applied perspective, a narrow host range makes specialist pathogens interesting for targeting specific pest insects since they have minimal direct effects on non-target species. Entomopathogenic fungi of the...

  11. A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

    DEFF Research Database (Denmark)

    Götz, C; Koenig, M G; Issinger, O G

    1995-01-01

    by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

  12. Molecular biology of baculovirus and its use in biological control in Brazil Biologia molecular de baculovírus e seu uso no controle biológico de pragas no Brasil

    Directory of Open Access Journals (Sweden)

    Maria Elita Batista de Castro

    1999-10-01

    Full Text Available Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses have polyhedrical inclusion bodies (PIBs containing multiple viral particles, while GVs (Granuloviruses appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus and PDV (Polyhedra Derived Virus, which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.Os baculovírus são vírus patogênicos a insetos, encontrados principalmente na ordem Lepidoptera. A família Baculoviridae é taxonomicamente dividida em dois gêneros: Nucleopolyhedrovirus e Granulovirus, que diferem pela morfologia do corpo de oclusão. Os nucleopoliedrovírus (NPV possuem corpos de inclusão poliédrica (PIB, contendo múltiplas partículas virais, enquanto os granulovírus (GV contêm, em geral, partículas únicas, ocluídas em corpos protéicos de forma ovóide. Durante o ciclo de vida são produzidos dois tipos de progênies virais: BV ("Budded Virus" e PDV ("Polyhedra Derived Virus", que são essenciais para o processo de infecção e propagação do vírus. Os baculovírus têm sido empregados no controle de pragas e, por serem específicos e restritos a invertebrados, s

  13. Insect pathogens as biological control agents: Back to the future.

    Science.gov (United States)

    Lacey, L A; Grzywacz, D; Shapiro-Ilan, D I; Frutos, R; Brownbridge, M; Goettel, M S

    2015-11-01

    The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens

  14. Development Of PIXE Measurement Of Ca Changes Resulting From Viral Transduction In Cells

    Science.gov (United States)

    Whitlow, Harry J.; Chienthavorn, Orapin; Eronen, Hannele; Sajavaara, Timo; Laitinen, Mikko; Norarat, Rattanaporn; Gilbert, Leona K.

    2011-06-01

    Ca is a life-element of particular interest because it is both bound to proteins, and as Ca2+ which functions as a signal molecule in apoptosis. Here we report development of chemical-matrix blind assaying the Ca fluxes from transduced HepG2 cells using particle induced X-ray emission. The cells were transduced with recombinant baculoviruses hosting the DNA for non-structural protein 1 (NS1) of the human pavovirus B19. Different recombinant baculoviruses were used that carried different DNA payloads of this NS1. Two different approaches have been developed to assay Ca in cells. The first is where the cells were directly cultured using a self-supporting pioloform as a substrate. In the second approach the cells are permeabilized, and bound-Ca content in the debris, and unbound-Ca in the wash solutions were measured using an internal V reference standard. The results support a difference in the Ca contents depending on the payload of the infecting virus, however the PIXE signals were too close to the minimum detection limit to draw reliable conclusions.

  15. Molecular Genetics of Beauveria bassiana Infection of Insects.

    Science.gov (United States)

    Ortiz-Urquiza, A; Keyhani, N O

    2016-01-01

    Research on the insect pathogenic filamentous fungus, Beauveria bassiana has witnessed significant growth in recent years from mainly physiological studies related to its insect biological control potential, to addressing fundamental questions regarding the underlying molecular mechanisms of fungal development and virulence. This has been in part due to a confluence of robust genetic tools and genomic resources for the fungus, and recognition of expanded ecological interactions with which the fungus engages. Beauveria bassiana is a broad host range insect pathogen that has the ability to form intimate symbiotic relationships with plants. Indeed, there is an increasing realization that the latter may be the predominant environmental interaction in which the fungus participates, and that insect parasitism may be an opportunist lifestyle evolved due to the carbon- and nitrogen-rich resources present in insect bodies. Here, we will review progress on the molecular genetics of B. bassiana, which has largely been directed toward identifying genetic pathways involved in stress response and virulence assumed to have practical applications in improving the insect control potential of the fungus. Important strides have also been made in understanding aspects of B. bassiana development. Finally, although increasingly apparent in a number of studies, there is a need for progressing beyond phenotypic mutant characterization to sufficiently investigate the molecular mechanisms underlying B. bassiana's unique and diverse lifestyles as saprophyte, insect pathogen, and plant mutualist. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Microbial Pest Control Agents: Are they a Specific And Safe Tool for Insect Pest Management?

    Science.gov (United States)

    Deshayes, Caroline; Siegwart, Myriam; Pauron, David; Froger, Josy-Anne; Lapied, Bruno; Apaire-Marchais, Véronique

    2017-01-01

    Microorganisms (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA). They are used as alternative strategies to chemical insecticides to counteract the development of resistances and to reduce adverse effects on both environment and human health. These natural entomopathogenic agents, which have specific modes of action, are generally considered safer as compared to conventional chemical insecticides. Baculoviruses are the only viruses being used as the safest biological control agents. They infect insects and have narrow host ranges. Bacillus thuringiensis (Bt) is the most widely and successfully used bioinsecticide in the integrated pest management programs in the world. Bt mainly produces crystal delta-endotoxins and secreted toxins. However, the Bt toxins are not stable for a very long time and are highly sensitive to solar UV. So genetically modified plants that express toxins have been developed and represent a large part of the phytosanitary biological products. Finally, entomopathogenic fungi and particularly, Beauveria bassiana and Metarhizium anisopliae, are also used for their insecticidal properties. Most studies on various aspects of the safety of MPCA to human, non-target organisms and environment have only reported acute but not chronic toxicity. This paper reviews the modes of action of MPCA, their toxicological risks to human health and ecotoxicological profiles together with their environmental persistence. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity". Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.

    Science.gov (United States)

    Shastry, Barkur S; Trese, Michael T

    2003-12-05

    Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography

  18. Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

    Science.gov (United States)

    Iatrou, K; Meidinger, R G

    1990-01-01

    A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

  19. Identification of Sumoylated Proteins in the Silkworm Bombyx mori

    Science.gov (United States)

    Tang, Xudong; Fu, Xuliang; Hao, Bifang; Zhu, Feng; Xiao, Shengyan; Xu, Li; Shen, Zhongyuan

    2014-01-01

    Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori. PMID:25470021

  20. Review of Vaccinia Virus and Baculovirus Viability Versus Virucides

    Science.gov (United States)

    2008-03-01

    25 6.4 Lignin ......................................................................................... 25 6.5...a lower pH (4.83 - 5.22), the virus rapidly inactivated over a month (Tomas et al., 1973). 16 The effects of alkalis on baculoviruses are important...of antioxidant and oxidative enzymes on UV inactivation by inhibiting the generation of highly reactive free radicals within HzSNPV. Water suspensions

  1. Biology and Genomics of Viruses Within the Genus Gammabaculovirus

    Directory of Open Access Journals (Sweden)

    Shannon Escasa

    2011-11-01

    Full Text Available Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

  2. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    Directory of Open Access Journals (Sweden)

    Roberto Franco Teixeira Corrêa

    Full Text Available Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ, resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7 when tested at 20 µg/mL.

  3. Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus

    Science.gov (United States)

    Clavijo, Gabriel; Williams, Trevor; Muñoz, Delia; Caballero, Primitivo; López-Ferber, Miguel

    2010-01-01

    An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission. PMID:19939845

  4. Natural Variation in Resistance to Virus Infection in Dipteran Insects

    Directory of Open Access Journals (Sweden)

    William H. Palmer

    2018-03-01

    Full Text Available The power and ease of Drosophila genetics and the medical relevance of mosquito-transmitted viruses have made dipterans important model organisms in antiviral immunology. Studies of virus–host interactions at the molecular and population levels have illuminated determinants of resistance to virus infection. Here, we review the sources and nature of variation in antiviral immunity and virus susceptibility in model dipteran insects, specifically the fruit fly Drosophila melanogaster and vector mosquitoes of the genera Aedes and Culex. We first discuss antiviral immune mechanisms and describe the virus-specificity of these responses. In the following sections, we review genetic and microbiota-dependent variation in antiviral immunity. In the final sections, we explore less well-studied sources of variation, including abiotic factors, sexual dimorphism, infection history, and endogenous viral elements. We borrow from work on other pathogen types and non-dipteran species when it parallels or complements studies in dipterans. Understanding natural variation in virus–host interactions may lead to the identification of novel restriction factors and immune mechanisms and shed light on the molecular determinants of vector competence.

  5. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

    DEFF Research Database (Denmark)

    Sørensen, K.J.; Madsen, K.G.; Madsen, E.S.

    1998-01-01

    The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after...... experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins....... The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave...

  6. Feasibility of a novel positive feedback effect of 131I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

    International Nuclear Information System (INIS)

    Guo Rui; Tian Lipeng; Han Bing; Xu Haoping; Zhang Miao; Li Biao

    2011-01-01

    Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by 131 I and may promote human NIS (hNIS) expression, hNIS also induces 131 I uptake and activates Egr1, so the existence of a positive feedback effect of 131 I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test 131 I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without 131 I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of 131 I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without 131 I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with 131 I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of 131 I after 131 I stimulation. The amount of 131 I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the 131 I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of 131 I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher 131 I uptake. It might provide a reference for the existence of a positive feedback effect in 131 I-promoted Bac-Egr1-h

  7. Interactions between parasites and insects vectors

    Directory of Open Access Journals (Sweden)

    Hilary Hurd

    1994-01-01

    Full Text Available This review stresses the importance of studies that will provide a basic understanding of the pathology of parasite-infected vector insects. This knowledge should be a vital component of the very focussed initiatives currently being funded in the areas of vector control. Vector fecundity reduction is discussed as an example of such pathology. Underlying mechanisms are being investigated in a model system, Hymenolepis diminuta-infected Tenebrio molitor and in Onchocerca-infected blackflies and Plasmodium-infected Anopheles stephensi. In all cases, host vitellogenesis is disrupted by the parasite and, in the tapeworm/beetle model, interaction between the parasite and the endocrine control of the insect's reproductive physiology has been demonstrated.

  8. Induced release of a plant-defense volatile 'deceptively' attracts insect vectors to plants infected with a bacterial pathogen.

    Directory of Open Access Journals (Sweden)

    Rajinder S Mann

    Full Text Available Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las, affects host preference behavior of its psyllid (Diaphorina citri Kuwayama vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of

  9. Continuous Influx of Genetic Material from Host to Virus Populations.

    Directory of Open Access Journals (Sweden)

    Clément Gilbert

    2016-02-01

    Full Text Available Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86 can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69 belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

  10. Assessing gene expression during pathogenesis: Use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans.

    Science.gov (United States)

    Lobo, Luciana S; Luz, Christian; Fernandes, Éverton K K; Juárez, M Patricia; Pedrini, Nicolás

    2015-06-01

    Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Insect cadaver applications: pros and cons

    Science.gov (United States)

    Application of entomopathogenic nematodes (EPNs) formulated as insect cadavers has become an alternative to aqueous application for the control of agricultural pests. In this approach, the infected insect host cadaver is applied directly to the target site and pest suppression is achieved by the inf...

  12. Immune responses to baculovirus-displayed enterovirus 71 VP1 antigen.

    Science.gov (United States)

    Kiener, Tanja K; Premanand, Balraj; Kwang, Jimmy

    2013-04-01

    The increased distribution and neurovirulence of enterovirus 71 is an important health threat for young children in Asia Pacific. Vaccine design has concentrated on inactivated virus with the most advanced undergoing Phase III clinical trials. By using a subunit vaccine approach, production costs could be reduced by lowering the need for biocontainment. In addition, novel mutations could be rapidly incorporated to reflect the emergence of new enterovirus 71 subgenogroups. To circumvent the problems associated with conventional subunit vaccines, the antigen can be displayed on a viral vector that conveys stability and facilitates purification. Additional advantages of viral-vectored subunit vaccines are their ability to stimulate the innate immune system by transducing cells and the possibility of oral or nasal delivery, which dispenses with the need for syringes and medical personnel. Baculovirus-displayed VP1 combines all these benefits with protection that is as efficient as inactivated virus.

  13. Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

    Science.gov (United States)

    Whitehill, Justin G A; Henderson, Hannah; Schuetz, Mathias; Skyba, Oleksandr; Yuen, Macaire Man Saint; King, John; Samuels, A Lacey; Mansfield, Shawn D; Bohlmann, Jörg

    2016-08-01

    Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. © 2015 John Wiley & Sons Ltd.

  14. Both cell-autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects.

    Science.gov (United States)

    Bear, Ashley; Monteiro, Antónia

    2013-08-01

    The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sex-specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals. © 2013 The Authors. BioEssays published by WILEY Periodicals, Inc.

  15. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    International Nuclear Information System (INIS)

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-01-01

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  16. Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus

    Directory of Open Access Journals (Sweden)

    Chie Goto

    2015-03-01

    Full Text Available Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV, and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%. In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.

  17. Number of infection events per cell during HIV-1 cell-free infection.

    Science.gov (United States)

    Ito, Yusuke; Remion, Azaria; Tauzin, Alexandra; Ejima, Keisuke; Nakaoka, Shinji; Iwasa, Yoh; Iwami, Shingo; Mammano, Fabrizio

    2017-07-26

    HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

  18. Several domains from VAR2CSA can induce Plasmodium falciparum adhesion-blocking antibodies

    DEFF Research Database (Denmark)

    Salanti, Ali; Resende, Mafalda; Ditlev, Sisse B

    2010-01-01

    . In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity. METHODS: All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested...

  19. Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

    Science.gov (United States)

    Mann, Rajinder S.; Ali, Jared G.; Hermann, Sara L.; Tiwari, Siddharth; Pelz-Stelinski, Kirsten S.; Alborn, Hans T.; Stelinski, Lukasz L.

    2012-01-01

    Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace

  20. Insect immunology and hematopoiesis

    OpenAIRE

    Hillyer, Julián F.

    2015-01-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and...

  1. Bovine respiratory syncytial virus: first serological evidence in Uruguay.

    Science.gov (United States)

    Costa, M; García, L; Yunus, A S; Rockemann, D D; Samal, S K; Cristina, J

    2000-01-01

    Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves resulting in a substantial economic loss for the cattle industry worldwide. In order to determine the presence of BRSV in Uruguay, an immunoenzymatic test was set up, using a recombinant BRSV nucleocapsid (N) protein as the antigen. The N protein was produced in Sf9 insect cells by a recombinant baculovirus expressing the N protein. Serum samples collected from one hundred cattle from four different geographic regions of Uruguay were analyzed. Antibodies against the N protein of BRSV were detected in 95% of the serum samples analyzed. These results show for the first time the presence of BRSV antibodies and suggest a widespread BRSV infection in the cattle population of Uruguay.

  2. Sunlight stability and rain-fastness of formulations of Baculovirus heliothis

    International Nuclear Information System (INIS)

    Ignoffo, C.M.; Garcia, C.; Saathoff, S.G.

    1997-01-01

    Sunlight-Ultraviolet, with an activity spectrum from 290 to 400 nm, is the most destructive factor affecting the persistence of baculoviruses. Benzopurpurin (a disazo dye) and carbon provided the best protection when polyhedral inclusion bodies (PIB) of Baculovirus heliothis were exposed to an artificial spectrum simulating sunlight-UV (UV). Greater than 75% of the original PIB activity was still present after 48 h of sunlight-UV. When sprayed on soybeans and exposed to natural sunlight, only formulations with carbon provided significant protection of PIB. The half-life of formulations were PIB-only 4.9 +/- 1.4 h (mean +/- SE), PIB + polymer (pyrrolidone-based sticker) 3.3 +/- 0.6 h, PIB + polymer + benzopurpurin 3.4 +/- 0.7 h, and PIB + polymer + carbon 27.7 +/- 5.2 h. PIB of B. heliothis tenaciously adhere to soybean, Glycine max (L.) Merrill, leaflets after spraying and drying. Less than 6% of the PIB activity of nonformulated PIB was lost after a drenching, simulated rainfall. More than 97% of the original PIB activity of carbon formulations was still present on soybean leaflets after 10 h of exposure to sunlight-UV. In contrast, 20% was present for formulations without carbon

  3. Mediator-Dependent Transcriptional Activation by Estrogen Receptor Bound to Distal Enhancers

    Science.gov (United States)

    2015-06-01

    TFIIE and TFIIF were expressed in bacteria and purified as described before34. Baculovirus-expressed TFIIA was purified from insect cells, as were...The spray voltage was set at 1.9–2.2 kV. The capillary temperature was 275 °C, and ion transmission on Velos S lenses was set at 35%. The instrument

  4. Inactivation of certain insect pathogens by ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Krieg, A.; Groener, A.; Huber, J.; Zimmermann, G.

    1981-01-01

    The UV-sensitivity of two baculoviruses (granulosis virus, nuclear polyhedrosis virus) and two entomopathogenic microorganisms (Bacillus thuringiensis, Beauveria bassiana) was determined by radiation tests. In the far UV (254 nm) the stability, measured at an inactivation rate of 99%, was in declining order: nuclear polyhedra >= conidia of B. bassiana > granula > spores of B. thuringiensis >= vegetative cells of B. thuringiensis. In the near UV (285-380 nm) the following order could be found: conidia of B. bassiana >= nuclear polyhedra > spores of B. thuringiensis >= granula > vegetative cells of B. thuringiensis. Far UV had a much higher germicidal effect for all pathogens tested than near UV.

  5. Combining the Sterile Insect Technique with the Incompatible Insect Technique: III-Robust Mating Competitiveness of Irradiated Triple Wolbachia-Infected Aedes albopictus Males under Semi-Field Conditions.

    Science.gov (United States)

    Zhang, Dongjing; Lees, Rosemary Susan; Xi, Zhiyong; Bourtzis, Kostas; Gilles, Jeremie R L

    2016-01-01

    Combination of the sterile insect technique with the incompatible insect technique is considered to be a safe approach to control Aedes albopictus populations in the absence of an accurate and scalable sex separation system or genetic sexing strain. Our previous study has shown that the triple Wolbachia-infected Ae. albopictus strain (wAlbA, wAlbB and wPip) was suitable for mass rearing and females could be completely sterilized as pupae with a radiation dose of at least 28 Gy. However, whether this radiation dose can influence the mating competitiveness of the triple infected males was still unknown. In this study we aimed to evaluate the effects of irradiation on the male mating competitiveness of the triple infected strain under laboratory and semi-field conditions. The results herein indicate that irradiation with a lower, female-sterilizing dose has no negative impact on the longevity of triple infected males while a reduced lifespan was observed in the wild type males (wAlbA and wAlbB) irradiated with a higher male-sterilizing dose, in small cages. At different sterile: fertile release ratios in small cages, triple-infected males induced 39.8, 81.6 and 87.8% sterility in a wild type female population at 1:1, 5:1 and 10:1 release ratios, respectively, relative to a fertile control population. Similarly, irradiated triple infected males induced 31.3, 70.5 and 89.3% sterility at 1:1, 5:1 and 10:1 release ratios, respectively, again relative to the fertile control. Under semi-field conditions at a 5:1 release ratio, relative to wild type males, the mean male mating competitiveness index of 28 Gy irradiated triple-infected males was significantly higher than 35 Gy irradiated wild type males, while triple infected males showed no difference in mean mating competitiveness to either irradiated triple-infected or irradiated wild type males. An unexpected difference was also observed in the relative male mating competitiveness of the triple infected strain after

  6. Toxicity of the mycotoxin fumonisin B1 on the insect Sf9 cell line.

    Science.gov (United States)

    Zhang, He; Zhang, Liyang; Diao, Xue; Li, Na; Liu, Chenglan

    2017-04-01

    Fumonisins are a type of mycotoxin produced by Fusarium spp., mainly F. proliferatum and F. vertieilliodes, and represent a potential hazard to the health of animals and human beings. The toxicity and mechanism of action of fumonisins is ambiguous, and it is unclear whether fumonisins are toxic to insect cells. This study examines the toxicity of fumonisin B 1 (FB 1 ) and its mechanism of action in the Spodoptera frugiperda Sf9 cell line. We found that FB 1 inhibited Sf9 cellular proliferation and arrested cell growth at the G 2 /M phase. Morphological observation showed that FB 1 induced swelling, vacuole formation, and loss of adhesion in Sf9 cells. Flow cytometry analysis showed that FB 1 caused depolarization of the cell membrane potential and hyperpolarization of the mitochondrial membrane potential. To uncover potential genes associated with the molecular mechanisms of FB 1 , 41 differentially expressed genes were identified by transcriptome analyses after FB 1 treatment. These genes are putatively involved in detoxification metabolism, insect hormone regulation, cell apoptosis, and other related processes. Finally, six differentially expressed genes were chosen and validated by quantitative real-time PCR (QRT-PCR). Our test could provide a reference for other kinds of insect cells studies on FB 1 stress. At the same time, our studies try to provide a possible for FB 1 as a precursor compounds of biological insecticide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    Science.gov (United States)

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  8. MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

    Science.gov (United States)

    Gradia, Scott D; Ishida, Justin P; Tsai, Miaw-Sheue; Jeans, Chris; Tainer, John A; Fuss, Jill O

    2017-01-01

    Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done. © 2017 Elsevier Inc. All rights reserved.

  9. Fungal endophytes which invade insect galls: insect pathogens, benign saprophytes, or fungal inquilines?

    Science.gov (United States)

    Wilson, Dennis

    1995-08-01

    Fungi are frequently found within insect galls. However, the origin of these fungi, whether they are acting as pathogens, saprophytes invading already dead galls, or fungal inquilines which invade the gall but kill the gall maker by indirect means, is rarely investigated. A pathogenic role for these fungi is usually inferred but never tested. I chose the following leaf-galling-insect/host-plant pairs (1) a cynipid which forms two-chambered galls on the veins of Oregon white oak, (2) a cynipid which forms single-chambered galls on California coast live oak, and (3) an aphid which forms galls on narrowleaf cottonwood leaves. All pairs were reported to have fungi associated with dead insects inside the gall. These fungi were cultured and identified. For the two cynipids, all fungi found inside the galls were also present in the leaves as fungal endophytes. The cottonwood leaves examined did not harbor fungal endophytes. For the cynipid on Oregon white oak, the fungal endophyte grows from the leaf into the gall and infects all gall tissue but does not directly kill the gall maker. The insect dies as a result of the gall tissue dying from fungal infection. Therefore, the fungus acts as an inquiline. Approximately 12.5% of these galls die as a result of invasion by the fungal endophyte.

  10. The effect of Beauveria bassiana infection on cell mediated and humoral immune response in house fly, Musca domestica L.

    Science.gov (United States)

    Mishra, Sapna; Kumar, Peeyush; Malik, Anushree

    2015-10-01

    Entomopathogenic fungi that manifest infections by overcoming insect's immune response could be a successful control agent for the house fly, Musca domestica L. which is a major domestic, medical, and veterinary pest. In this study, the immune response of house fly to Beauveria bassiana infection was investigated to reveal fundamental aspects of house fly hemocyte biology, such as hemocyte numbers and size, which is poorly understood. The total hemocyte counts (THCs) in B. bassiana-infected house fly showed an initial increase (from 6 to 9 h), followed by subsequent decrease (9 to 12 h) with increase in time of infection. The THCs was slightly greater in infected flies than the non-infected ones. Insight into relative hemocyte counts depicted a significant increase in prohemocyte (PR) and decrease in granulocyte (GR) in infected house flies compared to non-infected ones. The relative cell area of hemocyte cells showed a noticeable increase in PR and intermediate cells (ICs), while a considerable reduction was observed for plasmatocyte (PL) and GR. The considerable variation in relative cell number and cell area in the B. bassiana-infected house flies indicated stress development during infection. The present study highlights changes occurring during B. bassiana invasion to house fly leading to establishment of infection along with facilitation in understanding of basic hemocyte biology. The results of the study is expected to help in better understanding of house fly immune response during fungal infection, so as to assist production of more efficient mycoinsecticides for house fly control using B. bassiana.

  11. Identification of genes involved in DNA replication of the Autographa californica baculovirus

    NARCIS (Netherlands)

    Kool, M.; Ahrens, C. H.; Goldbach, R. W.; Rohrmann, G. F.; Vlak, J. M.

    1994-01-01

    By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2,

  12. Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.

    Science.gov (United States)

    Shokal, Upasana; Yadav, Shruti; Atri, Jaishri; Accetta, Julia; Kenney, Eric; Banks, Katherine; Katakam, Akash; Jaenike, John; Eleftherianos, Ioannis

    2016-02-09

    Symbiotic interactions between microbes and animals are common in nature. Symbiotic organisms are particularly common in insects and, in some cases, they may protect their hosts from pathogenic infections. Wolbachia and Spiroplasma endosymbionts naturally inhabit various insects including Drosophila melanogaster fruit flies. Therefore, this symbiotic association is considered an excellent model to investigate whether endosymbiotic bacteria participate in host immune processes against certain pathogens. Here we have investigated whether the presence of Wolbachia alone or together with Spiroplasma endosymbionts in D. melanogaster adult flies affects the immune response against the virulent insect pathogen Photorhabdus luminescens and against non-pathogenic Escherichia coli bacteria. We found that D. melanogaster flies carrying no endosymbionts, those carrying both Wolbachia and Spiroplasma, and those containing Wolbachia only had similar survival rates after infection with P. luminescens or Escherichia coli bacteria. However, flies carrying both endosymbionts or Wolbachia only contained higher numbers of E. coli cells at early time-points post infection than flies without endosymbiotic bacteria. Interestingly, flies containing Wolbachia only had lower titers of this endosymbiont upon infection with the pathogen P. luminescens than uninfected flies of the same strain. We further found that the presence of Wolbachia and Spiroplasma in D. melanogaster up-regulated certain immune-related genes upon infection with P. luminescens or E. coli bacteria, but it failed to alter the phagocytic ability of the flies toward E. coli inactive bioparticles. Our results suggest that the presence of Wolbachia and Spiroplasma in D. melanogaster can modulate immune signaling against infection by certain insect pathogenic and non-pathogenic bacteria. Results from such studies are important for understanding the molecular basis of the interactions between endosymbiotic bacteria of insects

  13. Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection.

    Science.gov (United States)

    Chen, Lin; Shen, Yunwang; Yang, Rui; Wu, Xiaofeng; Hu, Wenjun; Shen, Guoxin

    2015-10-24

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf64 (Bm64, a homologue of ac78) is a core baculovirus gene. Recently, Li et al. reported that Ac78 was not essential for budded viruses (BVs) production and occlusion-derived viruses (ODVs) formation (Virus Res 191:70-82, 2014). Conversely, Tao et al. demonstrated that Ac78 was localized to the BV and ODV envelopes and was required for BV production and ODV formation (J Virol 87:8441-50, 2013). In this study, the function of Bm64 was characterized to determine the role of Bm64 in the BmNPV infection cycle. The temporal expression of Bm64 was examined using total RNA extracted from BmNPV-infected BmN cells at different time points by reverse-transcription PCR (RT-PCR) and 5' RACE analysis. To determine the functions of Bm64 in viral replication and the viral phenotype throughout the viral life cycle, a deletion virus (vBm(64KO)) was generated via homologous recombination in Escherichia coli. Viral replication and BV production were determined by real-time PCR. Electron microscopy was used to detect virion morphogenesis. The subcellular localization of Bm64 was determined by microscopy, and per os infectivity was used to determine its role in the baculovirus oral infection cycle. Viral plaque and titer assay results showed that a few infectious BVs were produced by vBm(64KO), suggesting that deletion of Bm64 affected BV production. Viral DNA replication was detected and polyhedra were observed in vBm(64KO)-transfected cells. Microscopy analysis revealed that Bm64 was predominantly localized to the ring zone of the nuclei during the infection cycle. Electron microscopy showed that Bm64 was not essential for the formation of ODVs or the subsequent occlusion of ODV into polyhedra. The per os infectivity results showed that the polyhedra of vBm(64KO) were unable to infect silkworm larvae. In conclusion, our results suggest that Bm64 plays an important role in BV production and per os infection, but is not required for viral DNA

  14. Efferocytosis of Pathogen-Infected Cells

    Directory of Open Access Journals (Sweden)

    Niloofar Karaji

    2017-12-01

    Full Text Available The prompt and efficient clearance of unwanted and abnormal cells by phagocytes is termed efferocytosis and is crucial for organism development, maintenance of tissue homeostasis, and regulation of the immune system. Dying cells are recognized by phagocytes through pathways initiated via “find me” signals, recognition via “eat me” signals and down-modulation of regulatory “don’t eat me” signals. Pathogen infection may trigger cell death that drives phagocytic clearance in an immunologically silent, or pro-inflammatory manner, depending on the mode of cell death. In many cases, efferocytosis is a mechanism for eliminating pathogens and pathogen-infected cells; however, some pathogens have subverted this process and use efferocytic mechanisms to avoid innate immune detection and assist phagocyte infection. In parallel, phagocytes can integrate signals received from infected dying cells to elicit the most appropriate effector response against the infecting pathogen. This review focuses on pathogen-induced cell death signals that drive infected cell recognition and uptake by phagocytes, and the outcomes for the infected target cell, the phagocyte, the pathogen and the host.

  15. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    Science.gov (United States)

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Nitric oxide mediates insect cellular immunity via phospholipase A2 activation

    Science.gov (United States)

    After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Se...

  17. Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

    Science.gov (United States)

    Graham, L A; Bendena, W G; Walker, V K

    1996-02-01

    The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

  18. Inactivation of certain insect pathogens by ultraviolet radiation

    International Nuclear Information System (INIS)

    Krieg, A.; Groener, A.; Huber, J.; Zimmermann, G.

    1981-01-01

    The UV-sensitivity of two baculoviruses (granulosis virus, nuclear polyhedrosis virus) and two entomopathogenic microorganisms (Bacillus thuringiensis, Beauveria bassiana) was determined by radiation tests. In the far UV (254 nm) the stability, measured at an inactivation rate of 99%, was in declining order: nuclear polyhedra >= conidia of B. bassiana > granula > spores of B. thuringiensis >= vegetative cells of B. thuringiensis. In the near UV (285-380 nm) the following order could be found: conidia of B. bassiana >= nuclear polyhedra > spores of B. thuringiensis >= granula > vegetative cells of B. thuringiensis. Far UV had a much higher germicidal effect for all pathogens tested than near UV. (orig.) [de

  19. Biofuel cell backpacked insect and its application to wireless sensing.

    Science.gov (United States)

    Shoji, Kan; Akiyama, Yoshitake; Suzuki, Masato; Nakamura, Nobuhumi; Ohno, Hiroyuki; Morishima, Keisuke

    2016-04-15

    This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate β-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 μW (285 μW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NOVEL LIGNASE PROTEINS FROM TERMITES FOR DEPOLYMERIZATION OF LIGNOCELLULOSE

    Energy Technology Data Exchange (ETDEWEB)

    SLACK, JEFFREY, M.

    2012-12-06

    Wood is a potential source for biofuels such as ethanol if it can be digested into sugars and fermented by yeast. Biomass derived from wood is a challenging substrate for ethanol production since it is made of lignin and cellulose which cannot be broken down easily into fermentable sugars. Some insects, and termites in particular, are specialized at using enzymes in their guts to digest wood into sugars. If termite gut enzymes could be made abundantly by a recombinant protein expression vector system, they could be applied to an industrial process to make biofuels from wood. In this study, a large cDNA library of relevant termite genes was made using termites fed a normal diet, or a diet with added lignin. A subtracted library yielded genes that were overexpressed in the presence of lignin. Termite gut enzyme genes were identified and cloned into recombinant insect viruses called baculoviruses. Using our PERLXpress system for protein expression, these termite gene recombinant baculoviruses were prepared and used to infect insect larvae, which then expressed abundant recombinant termite enzymes. Many of these expressed enzymes were prepared to very high purity, and the activities were studied in conjunction with collaborators at Purdue University. Recombinant termite enzymes expressed in caterpillars were shown to be able to release sugars from wood. Mixing different combinations of these enzymes increased the amount of sugars released from a model woody biomass substrate. The most economical, fastest and energy conserving way to prepare termite enzymes expressed by recombinant baculoviruses in caterpillars was by making crude liquid homogenates. Making enzymes stable in homogenates therefore was a priority. During the course of these studies, improvements were made to the recombinant baculovirus expression platform so that caterpillar-derived homogenates containing expressed termite enzymes would be more stable. These improvements in the baculoviruses included

  1. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    Science.gov (United States)

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  2. Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

    Science.gov (United States)

    Pushparajan, Charlotte; Claus, Juan Daniel; Marshall, Sean D G; Visnovsky, Gabriel

    2017-12-01

    The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 10 5 cells ml -1 ) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 10 7 TCID 50 ml -1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm 2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

  3. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    International Nuclear Information System (INIS)

    McCarthy, Christina B.; Theilmann, David A.

    2008-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC ac142REP-ac143KO ). Fluorescence and light microscopy showed that infection by AcBAC ac142REP-ac143KO is limited to a single cell and titration assays confirmed that AcBAC ac142REP-ac143KO was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC ac142REP-ac143KO transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription

  4. Insect anaphylaxis: addressing clinical challenges.

    Science.gov (United States)

    Tracy, James M; Lewis, Elena J; Demain, Jeffrey G

    2011-08-01

    Few allergic reactions are as potentially life-threatening, or frightening to the patient, as anaphylaxis. Food, medications, and insect stings are the three most common triggers of anaphylaxis, but insect allergy provides the best opportunity to understand the biology of anaphylaxis. If the physician can establish a diagnosis of insect allergy, treatment with nearly 98% effectiveness can be initiated. However, sometimes patients have a compelling history of insect sting anaphylaxis, but negative skin and blood tests. This situation presents us with a fascinating opportunity to understand the biology of insect anaphylaxis. Recent and ongoing work shows that occult mast cell disease may be critical in insect anaphylaxis. Mastocytosis, serum tryptase and basophil biology are key elements; genetic markers may potentially help us diagnose at-risk individuals and determine proper treatment. Understanding basophil activation may play an additional role both in diagnosis and knowing when therapy might be terminated. Mast cell disease, serum tryptase and basophil biology are providing an opportunity to better understand and manage insect allergy. This evolving understanding should improve long-term management of insect anaphylaxis and help us to better understand the clinical dilemma of appropriate management of the history-positive patient in which testing is unable to detect venom-specific IgE. Furthermore, omalizumab's immunomodulatory effects may play a role in difficult-to-treat insect allergy and mastocytosis. Finally, unrelated to these, but still important as an ongoing risk factor, is the continued underutilization of epinephrine for both acute and long-term management of insect anaphylaxis.

  5. Nudiviruses and other large, double-stranded circular DNA viruses of invertebrates: new insights on an old topic.

    Science.gov (United States)

    Wang, Yongjie; Jehle, Johannes A

    2009-07-01

    Nudiviruses (NVs) are a highly diverse group of large, circular dsDNA viruses pathogenic for invertebrates. They have rod-shaped and enveloped nucleocapsids, replicate in the nucleus of infected host cells, and possess interesting biological and molecular properties. The unassigned viral genus Nudivirus has been proposed for classification of nudiviruses. Currently, the nudiviruses comprise five different viruses: the palm rhinoceros beetle virus (Oryctes rhinoceros NV, OrNV), the Hz-1 virus (Heliothis zea NV-1, HzNV-1), the cricket virus (Gryllus bimaculatus NV, GbNV), the corn earworm moth Hz-2 virus (HzNV-2), and the occluded shrimp Monodon Baculovirus reassigned as Penaeus monodon NV (PmNV). Thus far, the genomes of OrNV, GbNV, HzNV-1 and HzNV-2 have been completely sequenced. They vary between 97 and 230kbp in size and encode between 98 and 160 open reading frames (ORFs). All sequenced nudiviruses have 33 ORFs in common. Strikingly, 20 of them are homologous to baculovirus core genes involved in RNA transcription, DNA replication, virion structural components and other functions. Another nine conserved ORFs are likely associated with DNA replication, repair and recombination, and nucleotide metabolism; one is homologous to baculovirus iap-3 gene; two are nudivirus-specific ORFs of unknown function. Interestingly, one nudivirus ORF is similar to polh/gran gene, encoding occlusion body protein matrix and being conserved in Alpha- Beta- and Gammabaculoviruses. Members of nudiviruses are closely related and form a monophyletic group consisting of two sister clades of OrNV/GbNV and HzNVs/PmNV. It is proposed that nudiviruses and baculoviruses derived from a common ancestor and are evolutionarily related to other large DNA viruses such as the insect-specific salivary gland hypertrophy virus (SGHV) and the marine white spot syndrome virus (WSSV).

  6. Survival and Movement of Insect Parasitic Nematodes in Poultry Manure and Their Infectivity Against Musca domestica

    OpenAIRE

    Georgis, Ramon; Mullens, Bradley A.; Meyer, Jeffery A.

    1987-01-01

    Survival, infectivity, and movement of three insect parasitic nematodes (Steinernema feltiae All strain, S. bibionis SN strain, and Heterorhabditis heliothidis NC strain) in poultry manure were tested under laboratory conditions. The majority (70-100%) of the nematodes died within 18 hours after exposure to the manure. Nematodes exposed to manure slurry for 6 hours killed at least 95% of the house fly larvae, Musca domestica, but nematodes exposed for 12 hours achieved less than 40% larval mo...

  7. Expression of a male accessory gland peptide of Leptinotarsa decemlineata in insect cells infected with a recombinant baculovirus.

    NARCIS (Netherlands)

    Smid, H.M.; Schooneveld, H.; Deserno, M.L.L.G.; Put, B.; Vlak, J.M.

    1998-01-01

    The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8kDa peptide, designated Led-MAGP, that is recognized by monoclonal antibody MAC-18. The site of synthesis, amino acid sequence and the gene encoding this peptide have been documented (). The primary structure is homologous to

  8. Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus: A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection.

    Science.gov (United States)

    Wang, Guobao; Zhang, Jianjia; Shen, Yunwang; Zheng, Qin; Feng, Min; Xiang, Xingwei; Wu, Xiaofeng

    2015-06-01

    Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B. mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B. mori. Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

    Directory of Open Access Journals (Sweden)

    Yong Chen

    2017-11-01

    Full Text Available Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

  10. Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli.

    Science.gov (United States)

    Kueh, Chare Li; Yong, Chean Yeah; Masoomi Dezfooli, Seyedehsara; Bhassu, Subha; Tan, Soon Guan; Tan, Wen Siang

    2017-03-01

    Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017. © 2016 American Institute of Chemical Engineers.

  11. Expression of polyhedrin-hEGF fusion protein in cultured cells and ...

    African Journals Online (AJOL)

    For mass production of human epidermal growth factor (hEGF), silkworm baculovirus expression vector system (BEVS) was adopted in this study. hEGF gene was in-frame fused with polyhedrin (Ph) gene under the control of Ph promoter and was used to co-transfect BmN cell with the modified. Bombyx mori baculovirus ...

  12. Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria.

    Science.gov (United States)

    Bossard, Géraldine; Bartoli, Manon; Fardeau, Marie-Laure; Holzmuller, Philippe; Ollivier, Bernard; Geiger, Anne

    2017-09-03

    In humans, sleeping sickness (i.e. Human African Trypanosomiasis) is caused by the protozoan parasites Trypanosoma brucei gambiense (Tbg) in West and Central Africa, and T. b. rhodesiense in East Africa. We previously showed in vitro that Tbg is able to excrete/secrete a large number of proteins, including Translationally Controlled Tumor Protein (TCTP). Moreover, the tctp gene was described previously to be expressed in Tbg-infected flies. Aside from its involvement in diverse cellular processes, we have investigated a possible alternative role within the interactions occurring between the trypanosome parasite, its tsetse fly vector, and the associated midgut bacteria. In this context, the Tbg tctp gene was synthesized and cloned into the baculovirus vector pAcGHLT-A, and the corresponding protein was produced using the baculovirus Spodoptera frugicola (strain 9) / insect cell system. The purified recombinant protein rTbgTCTP was incubated together with bacteria isolated from the gut of tsetse flies, and was shown to bind to 24 out of the 39 tested bacteria strains belonging to several genera. Furthermore, it was shown to affect the growth of the majority of these bacteria, especially when cultivated under microaerobiosis and anaerobiosis. Finally, we discuss the potential for TCTP to modulate the fly microbiome composition toward favoring trypanosome survival.

  13. Insects as a Nitrogen Source for Plants

    Directory of Open Access Journals (Sweden)

    Michael J. Bidochka

    2013-07-01

    Full Text Available Many plants have evolved adaptations in order to survive in low nitrogen environments. One of the best-known adaptations is that of plant symbiosis with nitrogen-fixing bacteria; this is the major route by which nitrogen is incorporated into plant biomass. A portion of this plant-associated nitrogen is then lost to insects through herbivory, and insects represent a nitrogen reservoir that is generally overlooked in nitrogen cycles. In this review we show three specialized plant adaptations that allow for the recovery of insect nitrogen; that is, plants gaining nitrogen from insects. First, we show specialized adaptations by carnivorous plants in low nitrogen habitats. Insect carnivorous plants such as pitcher plants and sundews (Nepenthaceae/Sarraceniaceae and Drosera respectively are able to obtain substantial amounts of nitrogen from the insects that they capture. Secondly, numerous plants form associations with mycorrhizal fungi that can provide soluble nitrogen from the soil, some of which may be insect-derived nitrogen, obtained from decaying insects or insect frass. Finally, a specialized group of endophytic, insect-pathogenic fungi (EIPF provide host plants with insect-derived nitrogen. These soil-inhabiting fungi form a remarkable symbiosis with certain plant species. They can infect a wide range of insect hosts and also form endophytic associations in which they transfer insect-derived nitrogen to the plant. Root colonizing fungi are found in disparate fungal phylogenetic lineages, indicating possible convergent evolutionary strategies between taxa, evolution potentially driven by access to carbon-containing root exudates.

  14. Eicosanoid-mediated immunity in insects

    Science.gov (United States)

    Eicosanoid is a collective term for oxygenated metabolites of C20 polyunsaturated fatty acids. As seen in mammals, eicosanoids play crucial roles in mediating various physiological processes, including immune responses, in insects. Upon microbial pathogen infection, non-self recognition signals are ...

  15. Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection

    International Nuclear Information System (INIS)

    Barretto, Naina; Hallak, Louay K.; Peeples, Mark E.

    2003-01-01

    Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target

  16. Strep-Tagged Protein Purification.

    Science.gov (United States)

    Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

    2015-01-01

    The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

  17. Merkel cell polyomavirus infection and Merkel cell carcinoma.

    Science.gov (United States)

    Liu, Wei; MacDonald, Margo; You, Jianxin

    2016-10-01

    Merkel cell polyomavirus is the only polyomavirus discovered to date that is associated with a human cancer. MCPyV infection is highly prevalent in the general population. Nearly all healthy adults asymptomatically shed MCPyV from their skin. However, in elderly and immunosuppressed individuals, the infection can lead to a lethal form of skin cancer, Merkel cell carcinoma. In the last few years, new findings have established links between MCPyV infection, host immune response, and Merkel cell carcinoma development. This review discusses these recent discoveries on how MCPyV interacts with host cells to achieve persistent infection and, in the immunocompromised population, contributes to MCC development. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut.

    Directory of Open Access Journals (Sweden)

    Angela Kruse

    Full Text Available Huanglongbing, or citrus greening disease, is an economically devastating bacterial disease of citrus. It is associated with infection by the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP. For insect transmission to occur, CLas must be ingested during feeding on infected phloem sap and cross the gut barrier to gain entry into the insect vector. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome, respectively, of ACP gut tissue. CLas exposure resulted in changes in pathways involving the TCA cycle, iron metabolism, insecticide resistance and the insect's immune system. We identified 83 long non-coding RNAs that are responsive to CLas, two of which appear to be specific to the ACP. Proteomics analysis also enabled us to determine that Wolbachia, a symbiont of the ACP, undergoes proteome regulation when CLas is present. Fluorescent in situ hybridization (FISH confirmed that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia cells are prevalent throughout the gut epithelial cell cytoplasm, and Wolbachia titer is more variable in the guts of CLas exposed insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Our study provides a snapshot of how the psyllid gut copes with CLas exposure and provides information on pathways and proteins for targeted disruption of CLas-vector interactions at the gut interface.

  19. Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis.

    Science.gov (United States)

    Sugio, Akiko; Kingdom, Heather N; MacLean, Allyson M; Grieve, Victoria M; Hogenhout, Saskia A

    2011-11-29

    Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

  20. Developmental constraint of insect audition

    Directory of Open Access Journals (Sweden)

    Strauß Johannes

    2006-12-01

    Full Text Available Abstract Background Insect ears contain very different numbers of sensory cells, from only one sensory cell in some moths to thousands of sensory cells, e.g. in cicadas. These differences still await functional explanation and especially the large numbers in cicadas remain puzzling. Insects of the different orders have distinct developmental sequences for the generation of auditory organs. These sensory cells might have different functions depending on the developmental stages. Here we propose that constraints arising during development are also important for the design of insect ears and might influence cell numbers of the adults. Presentation of the hypothesis We propose that the functional requirements of the subadult stages determine the adult complement of sensory units in the auditory system of cicadas. The hypothetical larval sensory organ should function as a vibration receiver, representing a functional caenogenesis. Testing the hypothesis Experiments at different levels have to be designed to test the hypothesis. Firstly, the neuroanatomy of the larval sense organ should be analyzed to detail. Secondly, the function should be unraveled neurophysiologically and behaviorally. Thirdly, the persistence of the sensory cells and the rebuilding of the sensory organ to the adult should be investigated. Implications of the hypothesis Usually, the evolution of insect ears is viewed with respect to physiological and neuronal mechanisms of sound perception. This view should be extended to the development of sense organs. Functional requirements during postembryonic development may act as constraints for the evolution of adult organs, as exemplified with the auditory system of cicadas.

  1. HCV Infection and B-Cell Lymphomagenesis

    Directory of Open Access Journals (Sweden)

    Masahiko Ito

    2011-01-01

    Full Text Available Hepatitis C virus (HCV has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL. Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.

  2. Non-biting flying insects as carriers of pathogenic bacteria in a Brazilian hospital

    Directory of Open Access Journals (Sweden)

    Henrique Borges Kappel

    2013-04-01

    Full Text Available Introduction Insects have been described as mechanical vectors of nosocomial infections. Methods Non-biting flying insects were collected inside a pediatric ward and neonatal-intensive care unit (ICU of a Brazilian tertiary hospital. Results Most (86.4% of them were found to carry one or more species of bacteria on their external surfaces. The bacteria isolated were Gram-positive bacilli (68.2% or cocci (40.9%, and Gram-negative bacilli (18.2%. Conclusions Insects collected inside a hospital were carrying pathogenic bacteria; therefore, one must consider the possibility they may act as mechanical vectors of infections, in especially for debilitated or immune-compromised patients in the hospital environments where the insects were collected.

  3. Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

    Science.gov (United States)

    Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

    2008-10-01

    Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

  4. Isolation of a Spodoptera exigua baculovirus recombinant with retained biological activity despite a 10.6 kb deletion

    NARCIS (Netherlands)

    Dai, X.; Hajos, J.P.; Joosten, N.N.; Oers, van M.M.; IJkel, W.F.J.; Zuidema, D.; Yi, P.

    2000-01-01

    When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity

  5. Two Novel 30K Proteins Overexpressed in Baculovirus System and Their Antiapoptotic Effect in Insect and Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Wei Yu

    2013-01-01

    Full Text Available The 30K family of proteins is important in energy metabolism and may play a role in inhibiting cellular apoptosis in silkworms (Bombyx mori. Several 30K-family proteins have been identified. In this study, two new silkworm genes, referred to as Slp (NM 001126256 and Lsp-t (NM 001043443, were analyzed by a bioinformatics approach according to the sequences of 30K proteins previously reported in the silkworm. Both Slp and Lsp-t shared more than 41% amino acid sequence homology with the reported 30K proteins and displayed a conserved domain consistent with that of lipoprotein-11. Additionally, the cDNA sequences of both Slp and Lsp-t were obtained from the fat bodies of silkworm larvae by reverse transcription polymerase chain reaction. Both genes were expressed in BmN cells using the Bac-to-Bac system. Purified Slp and Lsp-t were added to cultured BmN and human umbilical vein endothelial cells (HUVEC that were treated with H2O2. Both Slp and Lsp-t significantly enhanced the viability and suppressed DNA fragmentation in H2O2 treated BmN and HUVEC cells. This study suggested that Slp and Lsp-t exhibit similar biological activities as their known 30K-protein counterparts and mediate an inhibitory effect against H2O2-induced apoptosis.

  6. SfDronc, an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda.

    Science.gov (United States)

    Huang, Ning; Civciristov, Srgjan; Hawkins, Christine J; Clem, Rollie J

    2013-05-01

    Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Subgenomic reporter RNA system for detection of alphavirus infection in mosquitoes.

    Directory of Open Access Journals (Sweden)

    J Jordan Steel

    Full Text Available Current methods for detecting real-time alphavirus (Family Togaviridae infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.

  8. Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

    Science.gov (United States)

    Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

    2017-05-25

    Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

  9. Genes involved in Beauveria bassiana infection to Galleria mellonella.

    Science.gov (United States)

    Chen, Anhui; Wang, Yulong; Shao, Ying; Zhou, Qiumei; Chen, Shanglong; Wu, Yonghua; Chen, Hongwei; Liu, Enqi

    2018-05-01

    The ascomycete fungus Beauveria bassiana is a natural pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. Many genes involved in fungal insecticide infection have been identified but few have been further explored. In this study, we constructed three transcriptomes of B. bassiana at 24, 48 and 72 h post infection of insect pests (BbI) or control (BbC). There were 3148, 3613 and 4922 genes differentially expressed at 24, 48 and 72 h post BbI/BbC infection, respectively. A large number of genes and pathways involved in infection were identified. To further analyze those genes, expression patterns across different infection stages (0, 12, 24, 36, 48, 60, 72 and 84 h) were studied using quantitative RT-PCR. This analysis showed that the infection-related genes could be divided into four patterns: highly expressed throughout the whole infection process (thioredoxin 1); highly expressed during early stages of infection but lowly expressed after the insect death (adhesin protein Mad1); lowly expressed during early infection but highly expressed after insect death (cation transporter, OpS13); or lowly expressed across the entire infection process (catalase protein). The data provide novel insights into the insect-pathogen interaction and help to uncover the molecular mechanisms involved in fungal infection of insect pests.

  10. Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

    Science.gov (United States)

    Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

    2017-02-01

    The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells

    DEFF Research Database (Denmark)

    Ozvegy, C.; Litman, Thomas; Szakacs, G.

    2001-01-01

    ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated...

  12. Structural Basis of the Interaction of a Trypanosoma cruzi Surface Molecule Implicated in Oral Infection with Host Cells and Gastric Mucin

    Science.gov (United States)

    Cortez, Cristian; Yoshida, Nobuko; Bahia, Diana; Sobreira, Tiago J.P.

    2012-01-01

    Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns Trypanosoma cruzi, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal β-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the α-helix. Located downstream and close to the α-helix was the gp82 gastric mucin binding site, which plays a central role in oral T. cruzi infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues. PMID:22860068

  13. Photodynamic and antibiotic therapy impair the pathogenesis of Enterococcus faecium in a whole animal insect model.

    Directory of Open Access Journals (Sweden)

    José Chibebe Junior

    Full Text Available Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS. PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics. In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively. In the study of antimicrobial PDT, we verified that methylene blue (MB injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192. Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095 or vancomycin treatment alone (P = 0.0025, suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to

  14. Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

    International Nuclear Information System (INIS)

    Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

    2003-01-01

    Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

  15. Evolutionary Ecology of Multitrophic Interactions between Plants, Insect Herbivores and Entomopathogens.

    Science.gov (United States)

    Shikano, Ikkei

    2017-06-01

    Plants play an important role in the interactions between insect herbivores and their pathogens. Since the seminal review by Cory and Hoover (2006) on plant-mediated effects on insect-pathogen interactions, considerable progress has been made in understanding the complexity of these tritrophic interactions. Increasing interest in the areas of nutritional and ecological immunology over the last decade have revealed that plant primary and secondary metabolites can influence the outcomes of insect-pathogen interactions by altering insect immune functioning and physical barriers to pathogen entry. Some insects use plant secondary chemicals and nutrients to prevent infections (prophylactic medication) and medicate to limit the severity of infections (therapeutic medication). Recent findings suggest that there may be selectable plant traits that enhance entomopathogen efficacy, suggesting that entomopathogens could potentially impose selection pressure on plant traits that improve both pathogen and plant fitness. Moreover, plants in nature are inhabited by diverse communities of microbes, in addition to entomopathogens, some of which can trigger immune responses in insect herbivores. Plants are also shared by numerous other herbivorous arthropods with different modes of feeding that can trigger different defensive responses in plants. Some insect symbionts and gut microbes can degrade ingested defensive phytochemicals and be orally secreted onto wounded plant tissue during herbivory to alter plant defenses. Since non-entomopathogenic microbes and other arthropods are likely to influence the outcomes of plant-insect-entomopathogen interactions, I discuss a need to consider these multitrophic interactions within the greater web of species interactions.

  16. Biogenic Amines in Insect Antennae

    Directory of Open Access Journals (Sweden)

    Marianna I. Zhukovskaya

    2017-06-01

    Full Text Available Insect antenna is a multisensory organ, each modality of which can be modulated by biogenic amines. Octopamine (OA and its metabolic precursor tyramine (TA affect activity of antennal olfactory receptor neurons. There is some evidence that dopamine (DA modulates gustatory neurons. Serotonin can serve as a neurotransmitter in some afferent mechanosensory neurons and both as a neurotransmitter and neurohormone in efferent fibers targeted at the antennal vessel and mechanosensory organs. As a neurohormone, serotonin affects the generation of the transepithelial potential by sensillar accessory cells. Other possible targets of biogenic amines in insect antennae are hygro- and thermosensory neurons and epithelial cells. We suggest that the insect antenna is partially autonomous in the sense that biologically active substances entering its hemolymph may exert their effects and be cleared from this compartment without affecting other body parts.

  17. Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection.

    Science.gov (United States)

    Wang, Qiang; Ju, Xiaoli; Chen, Liang; Chen, Keping

    2017-03-01

    Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

  18. Murid herpesvirus-4 exploits dendritic cells to infect B cells.

    Directory of Open Access Journals (Sweden)

    Miguel Gaspar

    2011-11-01

    Full Text Available Dendritic cells (DCs play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4, infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

  19. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  20. Alteration of cell cycle progression by Sindbis virus infection

    International Nuclear Information System (INIS)

    Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

    2015-01-01

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G 1 phase preferred to proliferate during S/G 2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G 1 phase than in cells infected during S/G 2 phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases

  1. Haemonchus contortus: Characterization of the baculovirus expressed form of aminopeptidase H11

    NARCIS (Netherlands)

    Reszka, N.; Rijsewijk, F.A.M.; Zelnik, V.; Moskwa, B.; Bienkowska-Szewczyk, K.

    2007-01-01

    Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned

  2. Multiorganismal insects: diversity and function of resident microorganisms.

    Science.gov (United States)

    Douglas, Angela E

    2015-01-07

    All insects are colonized by microorganisms on the insect exoskeleton, in the gut and hemocoel, and within insect cells. The insect microbiota is generally different from microorganisms in the external environment, including ingested food. Specifically, certain microbial taxa are favored by the conditions and resources in the insect habitat, by their tolerance of insect immunity, and by specific mechanisms for their transmission. The resident microorganisms can promote insect fitness by contributing to nutrition, especially by providing essential amino acids, B vitamins, and, for fungal partners, sterols. Some microorganisms protect their insect hosts against pathogens, parasitoids, and other parasites by synthesizing specific toxins or modifying the insect immune system. Priorities for future research include elucidation of microbial contributions to detoxification, especially of plant allelochemicals in phytophagous insects, and resistance to pathogens; as well as their role in among-insect communication; and the potential value of manipulation of the microbiota to control insect pests.

  3. Comparison of the Protective Efficacy of DNA and Baculovirus-Derived Protein Vaccines for EBOLA Virus in Guinea Pigs

    National Research Council Canada - National Science Library

    Mellquist-Riemenschneider, Jenny L; Garrison, Aura R; Geisbert, Joan B; Saikh, Kamal U; Heidebrink, Kelli D

    2003-01-01

    .... Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001...

  4. COMPARATIVE ASPECTS OF PLANT-CELL WALL DIGESTION IN INSECTS

    NARCIS (Netherlands)

    PRINS, RA; KREULEN, DA

    Although many phytophagous and wood-eating invertibrates form their own cellulases, there is an overwhelming variety of symbioses between plant- and wood-utilising insects and microorganisms. In one type of symbiosis (endosymbiosis), insects (rhinoceros beetle, cockroach, lower termites) host

  5. Behaviour of wild-type and genetically modified baculoviruses in the Helicoverpa armigera - cotton system: a simulation approach

    NARCIS (Netherlands)

    Sun, X.

    2005-01-01

    Keywords:   Helicoverpa armigera , baculovirus, genetic modification, cotton,transmission

  6. Phytoplasmas: bacteria that manipulate plants and insects.

    Science.gov (United States)

    Hogenhout, Saskia A; Oshima, Kenro; Ammar, El-Desouky; Kakizawa, Shigeyuki; Kingdom, Heather N; Namba, Shigetou

    2008-07-01

    Superkingdom Prokaryota; Kingdom Monera; Domain Bacteria; Phylum Firmicutes (low-G+C, Gram-positive eubacteria); Class Mollicutes; Candidatus (Ca.) genus Phytoplasma. Ca. Phytoplasma comprises approximately 30 distinct clades based on 16S rRNA gene sequence analyses of approximately 200 phytoplasmas. Phytoplasmas are mostly dependent on insect transmission for their spread and survival. The phytoplasma life cycle involves replication in insects and plants. They infect the insect but are phloem-limited in plants. Members of Ca. Phytoplasma asteris (16SrI group phytoplasmas) are found in 80 monocot and dicot plant species in most parts of the world. Experimentally, they can be transmitted by approximately 30, frequently polyphagous insect species, to 200 diverse plant species. In plants, phytoplasmas induce symptoms that suggest interference with plant development. Typical symptoms include: witches' broom (clustering of branches) of developing tissues; phyllody (retrograde metamorphosis of the floral organs to the condition of leaves); virescence (green coloration of non-green flower parts); bolting (growth of elongated stalks); formation of bunchy fibrous secondary roots; reddening of leaves and stems; generalized yellowing, decline and stunting of plants; and phloem necrosis. Phytoplasmas can be pathogenic to some insect hosts, but generally do not negatively affect the fitness of their major insect vector(s). In fact, phytoplasmas can increase fecundity and survival of insect vectors, and may influence flight behaviour and plant host preference of their insect hosts. The most common practices are the spraying of various insecticides to control insect vectors, and removal of symptomatic plants. Phytoplasma-resistant cultivars are not available for the vast majority of affected crops.

  7. Prostaglandins and their receptors in insect biology

    Directory of Open Access Journals (Sweden)

    David eStanley

    2011-12-01

    Full Text Available We treat the biological significance of prostaglandins (PGs and their known receptors in insect biology. PGs and related eicosanoids are oxygenated derivatives of arachidonic acid (AA and two other C20 polyunsaturated fatty acids. PGs are mostly appreciated in the context of biomedicine, but a growing body of literature indicates the biological significance of these compounds extends throughout the animal kingdom, and possibly beyond. PGs act in several crucial areas of insect biology. In reproduction, a specific PG, PGE2, releases oviposition behavior in most crickets and a few other insect species; PGs also mediate events in egg development in some species, which may represent all insects. PGs play major roles in modulating fluid secretion in Malpighian tubules, rectum and salivary glands, although, again, this has been studied in only a few insect species that may represent the Class. Insect immunity is a very complex defense system. PGs and other eicosanoids mediate a large number of immune reactions to infection and invasion. The actions of most PGs are mediated by specific receptors. Biomedical research has discovered a great deal of knowledge about PG receptors in mammals, including their structures, pharmacology, molecular biology and cellular locations. Studies of PG receptors in insects lag behind the biomedical background, however, recent results hold the promise of accelerated research in this area. A PG receptor has been identified in a class of lepidopteran hemocytes and experimentally linked to the release of prophenoloxidase. We conclude that research into PGs and their receptors in insects will lead to important advances in our understanding of insect biology.

  8. Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii

    International Nuclear Information System (INIS)

    Grujic, Ognjen; Grigg, Michael E.; Boulanger, Martin J.

    2008-01-01

    Preliminary X-ray diffraction studies of the bradyzoite-specific surface antigen BSR4 from T. gondii are described. Toxoplasma gondii is an important global pathogen that infects nearly one third of the world’s adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 Å resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4 1 2 1 2 or P4 3 2 1 2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 Å 3 Da −1

  9. Identification and characterization of an antennae-specific aldehyde oxidase from the navel orangeworm.

    Directory of Open Access Journals (Sweden)

    Young-Moo Choo

    Full Text Available Antennae-specific odorant-degrading enzymes (ODEs are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes--the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs. Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW, Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13-16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13-16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.

  10. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    LENUS (Irish Health Repository)

    Ryan, Ruth CM

    2011-10-24

    Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

  11. Baculovirus display of fusion protein of Peste des petits ruminants virus and hemagglutination protein of Rinderpest virus and immunogenicity of the displayed proteins in mouse model

    International Nuclear Information System (INIS)

    Masmudur Rahman, Md.; Shaila, M.S.; Gopinathan, Karumathil P.

    2003-01-01

    Recombinant Bombyx mori nucleopolyhedroviruses (BmNPV) displaying the immunodominant ectodomains of fusion glycoprotein (F) of Peste des petitis ruminants virus (PPRV) and the hemagglutinin protein (H) of Rinderpest virus (RPV), on the budded virions as well as the surface of the infected host cells have been constructed. The F and H protein sequences were inserted in-frame within the amino-terminal region of BmNPV envelope glycoprotein GP64 expressing under the strong viral polyhedrin (polh) promoter. We improved the recombinant virus selection in BmNPV by incorporating the green fluorescent protein gene (gfp) as selection marker under a separate promoter within the transfer cassette harboring the desired genes. Following infection of the insect larvae or the host-derived BmN cells with these recombinant BmNPVs, the expressed GP64 fusion proteins were displayed on the host cell surface and the budded virions. The antigenic epitopes of the recombinant proteins were properly displayed and the recombinant virus particles induced immune response in mice against PPRV or RPV

  12. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  13. Bimodal cell death induced by high radiation doses in the radioresistant sf9 insect cell line

    International Nuclear Information System (INIS)

    Chandna, S.

    2003-01-01

    Full text: This study was conducted to investigate the mode(s) of cell death induced by high radiation doses in the highly radioresistant Sf9 insect ovarian cell line. Methods: Cells were exposed to γ-radiation doses 200Gy and 500Gy, harvested at various time intervals (6h-72h) following irradiation, and subjected to cell morphology assay, DNA agarose gel electrophoresis, single cell gel electrophoresis (SCGE; comet assay) and Annexin-V labeling for the detection of membrane phosphatidylserine externalization. Cell morphology was assessed in cells entrapped and fixed in agarose gel directly from the cell suspension, thus preventing the possible loss of fragments/ apoptotic bodies. Surviving fraction of Sf9 cells was 0.01 at 200Gy and 98%) undergoing extensive DNA fragmentation at 500Gy, whereas the frequency of cells with DNA fragmentation was considerably less (∼12%) at 200Gy. Conclusions: While the mode of cell death at 200Gy seems to be different from typical apoptosis, a dose of 500Gy induced bimodal cell death, with typical apoptotic as well as the atypical cell death observed at 200Gy

  14. Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

    Science.gov (United States)

    Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

    2015-10-01

    The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Polypeptide synthesis in alphavirus-infected aedes albopictus cells during the establishment of persistent infection

    International Nuclear Information System (INIS)

    Richardson, M.A.; Boulton, R.W.; Raghow, R.S.; Dalgarno, L.

    1980-01-01

    Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cells, RRV reached peak titres at 34-48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was not shutdown of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s).The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9-11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity. (author)

  16. Environmental RNAi in herbivorous insects.

    Science.gov (United States)

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C; Johnson, Steven; Meyer, Steve E; Kerstetter, Randy A; McNulty, Brian C; Bolognesi, Renata; Heck, Gregory R

    2015-05-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. © 2015 Ivashuta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. ION-EXCHANGE IMMUNOAFFINITY PURIFICATION OF A RECOMBINANT BACULOVIRUS PLASMODIUM-FALCIPARUM APICAL MEMBRANE ANTIGEN, PF83/AMA-1

    NARCIS (Netherlands)

    NARUM, DL; WELLING, GW; THOMAS, AW

    1993-01-01

    A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83 000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-FG8-1. The

  18. Sterilizing insects with ionizing radiation

    International Nuclear Information System (INIS)

    Bakri, A.; Mehta, K.; Lance, D.R.

    2005-01-01

    Exposure to ionizing radiation is currently the method of choice for rendering insects reproductively sterile for area-wide integrated pest management (AW-IPM) programmes that integrate the sterile insect technique (SIT). Gamma radiation from isotopic sources (cobalt-60 or caesium-137) is most often used, but high-energy electrons and X-rays are other practical options. Insect irradiation is safe and reliable when established safety and quality-assurance guidelines are followed. The key processing parameter is absorbed dose, which must be tightly controlled to ensure that treated insects are sufficiently sterile in their reproductive cells and yet able to compete for mates with wild insects. To that end, accurate dosimetry (measurement of absorbed dose) is critical. Irradiation data generated since the 1950s, covering over 300 arthropod species, indicate that the dose needed for sterilization of arthropods varies from less than 5 Gy for blaberid cockroaches to 300 Gy or more for some arctiid and pyralid moths. Factors such as oxygen level, and insect age and stage during irradiation, and many others, influence both the absorbed dose required for sterilization and the viability of irradiated insects. Consideration of these factors in the design of irradiation protocols can help to find a balance between the sterility and competitiveness of insects produced for programmes that release sterile insects. Many programmes apply 'precautionary' radiation doses to increase the security margin of sterilization, but this overdosing often lowers competitiveness to the point where the overall induced sterility in the wild population is reduced significantly. (author)

  19. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

    Directory of Open Access Journals (Sweden)

    Mikaël Boullé

    2016-11-01

    Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

  20. Brucella abortus-infected B cells induce osteoclastogenesis.

    Science.gov (United States)

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2016-09-01

    Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. Infectivity of Oryctes Nudivirus Produced on Cell Culture Dsir Ha-1179 Against Larvae and Its Effects on Feeding of Neonates of Rhinoceros Beetle, Oryctes Rhinoceros

    International Nuclear Information System (INIS)

    Nur Ain Farhah Ros Saidon Khudri; Wahizatul Afzan Azmi; Ramle Moslim; Norman Kamarudin; Siti Ramlah Ahmad Ali

    2016-01-01

    The Oryctes nudivirus (OrNV) is a classical biocontrol agent for a major oil palm insect pest the rhinoceros beetle, Oryctes rhinoceros. The infectivity of three Malaysian indigenous types of OrNV types A, B and C were tested on larvae and neonates. On larvae, the peroral inoculation test technique indicated that the highest mortality of 100 % was achieved using type A produced from cell culture DSIR-HA-1179, while the highest infectivity of 41.7 % was recorded for type A prepared from infected guts. No differences in infectivity were observed on other treatments, which ranged from 13.1 % to 41.7 %. In the substrate contamination inoculation test technique, results showed that the level of infectivity was even lower in all OrNV treatments, ranging as low as 6.7 % to only 15.0 %. Low infectivity was mainly due to inactivation of virus inocula in the larval food substrates. Based on the results for both inoculation methods, the OrNV type C prepared from cell culture DSIR-HA-1179 was found more effective in controlling the L3 larvae than the other types of OrNV. The impact of OrNV infection on food consumption by the neonates was studied. The feeding of inoculated neonates with OrNV reduced rapidly, especially at the early stage of the experiment between eight days after treatment (DAT) to 16 DAT. At this period, the food consumption by all tested OrNV was rapidly reduced and maintained low until the experiment ended at 60 DAT. The highest feeding reduction rate was on neonates treated by type A (-0.074x) followed by neonates treated by type C (-0.053x) and type B (-0.035x). Therefore, it was suggested that besides on highly virulent, the selection of OrNV for field release should also based on high reduction rate on food consumption by the infected insects on plant hosts. (author)

  2. From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity.

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    Geraldine Mulley

    Full Text Available Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called "nutritional virulence" strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

  3. A Plant Bacterial Pathogen Manipulates Its Insect Vector's Energy Metabolism

    Science.gov (United States)

    Hijaz, Faraj; Ebert, Timothy A.; Rogers, Michael E.

    2016-01-01

    ABSTRACT Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, behavior, and metabolism. Because these pathogens interact with their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. “Candidatus Liberibacter asiaticus” (CLas) is transmitted in a persistent, circulative, and propagative manner. The genome of CLas revealed the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were significantly higher in CLas-infected than healthy psyllids. Gene expression analysis showed upregulation for ATP synthase subunits, while ATPase enzyme activity showed a decrease in ATPase activity. These results indicated that CLas stimulated Diaphorina citri to produce more ATP and many other energetic nucleotides, while it may inhibit their consumption by the insect. As a result of ATP accumulation, the adenylated energy charge (AEC) increased and the AMP/ATP and ADP/ATP ratios decreased in CLas-infected D. citri psyllids. Survival analysis confirmed a shorter life span for CLas-infected D. citri psyllids. In addition, electropenetrography showed a significant reduction in total nonprobing time, salivation time, and time from the last E2 (phloem ingestion) to the end of recording, indicating that CLas-infected psyllids were at a higher hunger level and they tended to forage more often. This increased feeding activity reflects the CLas-induced energetic stress. In conclusion, CLas alters the energy metabolism of its psyllid vector, D. citri, in order to secure its need for energetic nucleotides. IMPORTANCE Insect transmission of plant-pathogenic bacteria involves propagation and circulation of the bacteria within their vectors. The transmission process is complex and requires specific interactions at the molecular

  4. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    Energy Technology Data Exchange (ETDEWEB)

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.

    1986-06-01

    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

  5. Mast cells in viral infections

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    Piotr Witczak

    2012-04-01

    Full Text Available  There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9, but also RIG-I-like and NOD-like molecules. Furthermore, mast cells generate various mediators, cytokines and chemokines which modulate the intensity of inflammation and regulate the course of innate and adaptive anti-viral immunity. Indirect evidence for the role of mast cells in viral infections is also provided by clinical observations and results of animal studies. Currently, more and more data indicate that mast cells can be infected by some viruses (dengue virus, adenoviruses, hantaviruses, cytomegaloviruses, reoviruses, HIV-1 virus. It is also demonstrated that mast cells can release pre formed mediators as well as synthesize de novo eicosanoids in response to stimulation by viruses. Several data indicate that virus-stimulated mast cells secrete cytokines and chemokines, including interferons as well as chemokines with a key role in NK and Tc lymphocyte influx. Moreover, some information indicates that mast cell stimulation via TLR3, TLR7/8 and TLR9 can affect their adhesion to extracellular matrix proteins and chemotaxis, and influence expression of some membrane molecules. Critical analysis of current data leads to the conclusion that it is not yet possible to make definitive statements about the role of mast cells in innate and acquired defense mechanisms developing in the course of viral infection and/or pathomechanisms of viral diseases.

  6. Evolutionary conservation and changes in insect TRP channels.

    Science.gov (United States)

    Matsuura, Hironori; Sokabe, Takaaki; Kohno, Keigo; Tominaga, Makoto; Kadowaki, Tatsuhiko

    2009-09-10

    TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this

  7. Evolutionary conservation and changes in insect TRP channels

    Directory of Open Access Journals (Sweden)

    Tominaga Makoto

    2009-09-01

    Full Text Available Abstract Background TRP (Transient Receptor Potential channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. Results All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA. NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. Conclusion The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP

  8. History and Current Status of Development and Use of Viral Insecticides in China

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    Xiulian Sun

    2015-01-01

    Full Text Available The use of insect viruses as biological control agents started in the early 1960s in China. To date, more than 32 viruses have been used to control insect pests in agriculture, forestry, pastures, and domestic gardens in China. In 2014, 57 products from 11 viruses were authorized as commercial viral insecticides by the Ministry of Agriculture of China. Approximately 1600 tons of viral insecticidal formulations have been produced annually in recent years, accounting for about 0.2% of the total insecticide output of China. The development and use of Helicoverpa armigera nucleopolyhedrovirus, Mamestra brassicae nucleopolyhedrovirus, Spodoptera litura nucleopolyhedrovirus, and Periplaneta fuliginosa densovirus are discussed as case studies. Additionally, some baculoviruses have been genetically modified to improve their killing rate, infectivity, and ultraviolet resistance. In this context, the biosafety assessment of a genetically modified Helicoverpa armigera nucleopolyhedrovirus is discussed.

  9. When Eggs Don't Hatch. The Benefits of the Sterile Insect Technique

    International Nuclear Information System (INIS)

    Kilian, Lizette

    2012-01-01

    Insect pests, such as the medfly, tsetse flies and carob moth can devastate crops and infect herds, causing severe economic hardship. To suppress the insect pest population and protect their livestock and crops, farmers usually use large quantities of pesticides. However, these pesticides are expensive, a risk to public health and cause environmental damage. Another technique, however, can reduce the insect pest population using natural means that do not require toxic chemicals: the sterile insect technique, or SIT. When female insect pests mate with male partners that have been radiation sterilized, the insemination produces eggs that cannot hatch. Since mating does not produce offspring, the insect population decreases naturally. The pest population can be suppressed with little or no use of pesticides. With the help of the IAEA, farmers have applied SIT successfully in over 20 countries on five continents, for over 15 insect species worldwide.

  10. Within the fortress: A specialized parasite is not discriminated against in a social insect society.

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    Emilia Solá Gracia

    Full Text Available Social insect colonies function cohesively due, in part, to altruistic behaviors performed towards related individuals. These colonies can be affected by parasites in two distinct ways, either at the level of the individual or the entire colony. As such, colonies of social insects can experience conflict with infected individuals reducing the cohesiveness that typifies them. Parasites of social insects therefore offer us a framework to study conflicts within social insect colonies in addition to the traditionally viewed conflicts afforded by groups of low genetic relatedness due to multiple mating for example. In our study, we use the behavior manipulating fungal pathogen, Ophiocordyceps kimflemingiae (= unilateralis and its host, Camponotus castaneus, to ask if colony members are able to detect infected individuals. Such detection would be optimal for the colony since infected workers die near foraging trails where the fungus develops its external structures and releases spores that infect other colony members. To determine if C. castaneus workers can detect these future threats, we used continuous-time point observations coupled with longer continuous observations to discern any discrimination towards infected individuals. After observing 1,240 hours of video footage we found that infected individuals are not removed from the colony and continuously received food during the course of fungal infection. We also calculated the distances between workers and the nest entrance in a total of 35,691 data points to find infected workers spent more time near the entrance of the nest. Taken together, these results suggest healthy individuals do not detect the parasite inside their nestmates. The colony's inability to detect infected individuals allows O. kimflemingiae to develop within the colony, while receiving food and protection from natural enemies, which could damage or kill its ant host before the parasite has completed its development.

  11. Combination of anti-retroviral drugs and radioimmunotherapy specifically kills infected cells from HIV infected individuals

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    Dina Tsukrov

    2016-09-01

    Full Text Available Eliminating virally infected cells is an essential component of any HIV eradication strategy. Radioimmunotherapy (RIT, a clinically established method for killing cells using radiolabeled antibodies, was recently applied to target HIV-1 gp41 antigen expressed on the surface of infect-ed cells. Since gp41 expression by infected cells is likely down-regulated in patients on an-tiretroviral therapy (ART, we evaluated the ability of RIT to kill ART-treated infected cells us-ing both in vitro models and lymphocytes isolated from HIV-infected subjects. Human peripheral blood mononuclear cells (PBMCs were infected with HIV and cultured in the presence of two clinically relevant ART combinations. Scatchard analysis of the 2556 human monoclonal anti-body to HIV gp41 binding to the infected and ART-treated cells demonstrated sufficient residual expression of gp41 on the cell surface to warrant subsequent RIT. This is the first time the quantification of gp41 post-ART is being reported. Cells were then treated with Bismuth-213-labeled 2556 antibody. conjugated to the human monoclonal antibody 2556, which binds to HIV gp41. Cell survival was quantified by Trypan blue and residual viremia by p24 ELISA. Cell surface gp41 expression was assessed by Scatchard analysis. The experiments were repeated using PBMCs isolated from blood specimens obtained from 15 HIV-infected individuals: ten on ART and five ART-naive. We found that 213Bi-2556 killed ART-treated infected PBMCs and reduced viral production to undetectable levels. ART and RIT co-treatment was more effective at reducing viral load in vitro than either therapy alone, indicating that gp41 expression under ART was sufficient to allow 213Bi-2556 to deliver cytocidal doses of radiation to infected cells. This study provides proof of concept that 213Bi-2556 may represent an innovative and effective targeting method for killing HIV-infected cells treated with ART, and supports continued development of 213Bi

  12. The MrCYP52 cytochrome P450 monoxygenase gene of Metarhizium robertsii is important for utilizing insect epicuticular hydrocarbons.

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    Liangcai Lin

    Full Text Available Fungal pathogens of plants and insects infect their hosts by direct penetration of the cuticle. Plant and insect cuticles are covered by a hydrocarbon-rich waxy outer layer that represents the first barrier against infection. However, the fungal genes that underlie insect waxy layer degradation have received little attention. Here we characterize the single cytochrome P450 monoxygenase family 52 (MrCYP52 gene of the insect pathogen Metarhizium robertsii, and demonstrate that it encodes an enzyme required for efficient utilization of host hydrocarbons. Expressing a green florescent protein gene under control of the MrCYP52 promoter confirmed that MrCYP52 is up regulated on insect cuticle as well as by artificial media containing decane (C10, extracted cuticle hydrocarbons, and to a lesser extent long chain alkanes. Disrupting MrCYP52 resulted in reduced growth on epicuticular hydrocarbons and delayed developmental processes on insect cuticle, including germination and production of appressoria (infection structures. Extraction of alkanes from cuticle prevented induction of MrCYP52 and reduced growth. Insect bioassays against caterpillars (Galleria mellonella confirmed that disruption of MrCYP52 significantly reduces virulence. However, MrCYP52 was dispensable for normal germination and appressorial formation in vitro when the fungus was supplied with nitrogenous nutrients. We conclude therefore that MrCYP52 mediates degradation of epicuticular hydrocarbons and these are an important nutrient source, but not a source of chemical signals that trigger infection processes.

  13. Surviving embryogenesis : The extraembryonic serosa protects the insect egg against desiccation and infection

    NARCIS (Netherlands)

    Jacobs, Chris Gerardus Cornelus

    2014-01-01

    Insects are the most diverse group of animals on earth. They inhabit nearly all terrestrial habitats. One of the factors underlying this success is the ability of insect eggs to survive in adverse conditions. For a long time the ability to survive these adverse conditions has been attributed to

  14. Fungal cell gigantism during mammalian infection.

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    Oscar Zaragoza

    2010-06-01

    Full Text Available The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  15. Fungal cell gigantism during mammalian infection.

    Science.gov (United States)

    Zaragoza, Oscar; García-Rodas, Rocío; Nosanchuk, Joshua D; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

    2010-06-17

    The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  16. Brain transcriptomes of honey bees (Apis mellifera experimentally infected by two pathogens: Black queen cell virus and Nosema ceranae

    Directory of Open Access Journals (Sweden)

    Vincent Doublet

    2016-12-01

    Full Text Available Regulation of gene expression in the brain plays an important role in behavioral plasticity and decision making in response to external stimuli. However, both can be severely affected by environmental factors, such as parasites and pathogens. In honey bees, the emergence and re-emergence of pathogens and potential for pathogen co-infection and interaction have been suggested as major components that significantly impaired social behavior and survival. To understand how the honey bee is affected and responds to interacting pathogens, we co-infected workers with two prevalent pathogens of different nature, the positive single strand RNA virus Black queen cell virus (BQCV, and the Microsporidia Nosema ceranae, and explored gene expression changes in brains upon single infections and co-infections. Our data provide an important resource for research on honey bee diseases, and more generally on insect host-pathogen and pathogen-pathogen interactions. Raw and processed data are publicly available in the NCBI/GEO database: (http://www.ncbi.nlm.nih.gov/geo/ under accession number GSE81664.

  17. Characterizing Enterovirus 71 and Coxsackievirus A16 virus-like particles production in insect cells.

    Science.gov (United States)

    Somasundaram, Balaji; Chang, Cindy; Fan, Yuan Y; Lim, Pei-Yin; Cardosa, Jane; Lua, Linda

    2016-02-15

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five™ cells. Lowering the incubation temperature from the standard 27°C to 21°C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7× higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15±1nm and 15.3±5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa.

    Science.gov (United States)

    Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E

    2008-02-19

    Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.

  19. Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

    Science.gov (United States)

    Vertyporokh, Lidiia; Wojda, Iwona

    2017-01-01

    The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

  20. Intracellular Events and Cell Fate in Filovirus Infection

    Directory of Open Access Journals (Sweden)

    Elena Ryabchikova

    2011-08-01

    Full Text Available Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.

  1. Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

    International Nuclear Information System (INIS)

    Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

    2013-01-01

    Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication

  2. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs.

    Science.gov (United States)

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H; van Rij, Ronald P

    2016-02-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.

  3. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

    Science.gov (United States)

    Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

    2010-10-01

    Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

  4. NKT cell depletion in humans during early HIV infection.

    Science.gov (United States)

    Fernandez, Caroline S; Kelleher, Anthony D; Finlayson, Robert; Godfrey, Dale I; Kent, Stephen J

    2014-08-01

    Natural killer T (NKT) cells bridge across innate and adaptive immune responses and have an important role in chronic viral infections such as human immunodeficiency virus (HIV). NKT cells are depleted during chronic HIV infection, but the timing, drivers and implications of this NKT cell depletion are poorly understood. We studied human peripheral blood NKT cell levels, phenotype and function in 31 HIV-infected subjects not on antiretroviral treatment from a mean of 4 months to 2 years after HIV infection. We found that peripheral CD4(+) NKT cells were substantially depleted and dysfunctional by 4 months after HIV infection. The depletion of CD4(+) NKT cells was more marked than the depletion of total CD4(+) T cells. Further, the early depletion of NKT cells correlated with CD4(+) T-cell decline, but not HIV viral levels. Levels of activated CD4(+) T cells correlated with the loss of NKT cells. Our studies suggest that the early loss of NKT cells is associated with subsequent immune destruction during HIV infection.

  5. Parametric structural modeling of insect wings

    International Nuclear Information System (INIS)

    Mengesha, T E; Vallance, R R; Barraja, M; Mittal, R

    2009-01-01

    Insects produce thrust and lift forces via coupled fluid-structure interactions that bend and twist their compliant wings during flapping cycles. Insight into this fluid-structure interaction is achieved with numerical modeling techniques such as coupled finite element analysis and computational fluid dynamics, but these methods require accurate and validated structural models of insect wings. Structural models of insect wings depend principally on the shape, dimensions and material properties of the veins and membrane cells. This paper describes a method for parametric modeling of wing geometry using digital images and demonstrates the use of the geometric models in constructing three-dimensional finite element (FE) models and simple reduced-order models. The FE models are more complete and accurate than previously reported models since they accurately represent the topology of the vein network, as well as the shape and dimensions of the veins and membrane cells. The methods are demonstrated by developing a parametric structural model of a cicada forewing.

  6. Context dependency and generality of fever in insects

    Science.gov (United States)

    Stahlschmidt, Z. R.; Adamo, S. A.

    2013-07-01

    Fever can reduce mortality in infected animals. Yet, despite its fitness-enhancing qualities, fever often varies among animals. We used several approaches to examine this variation in insects. Texas field crickets ( Gryllus texensis) exhibited a modest fever (1 °C increase in preferred body temperature, T pref) after injection of prostaglandin, which putatively mediates fever in both vertebrates and invertebrates, but they did not exhibit fever during chronic exposure to heat-killed bacteria. Further, chronic food limitation and mating status did not affect T pref or the expression of behavioural fever, suggesting limited context dependency of fever in G. texensis. Our meta-analysis of behavioural fever studies indicated that behavioural fever occurs in many insects, but it is not ubiquitous. Thus, both empirical and meta-analytical results suggest that the fever response in insects `is widespread, although certainly not inevitable' (Moore 2002). We highlight the need for future work focusing on standardizing an experimental protocol to measure behavioural fever, understanding the specific mechanism(s) underlying fever in insects, and examining whether ecological or physiological costs often outweigh the benefits of fever and can explain the sporadic nature of fever in insects.

  7. A plant pathogenic bacterium exploits the tricarboxylic acid cycle metabolic pathway of its insect vector

    Science.gov (United States)

    Nehela, Yasser; Hijaz, Faraj; Vincent, Christopher I.

    2018-01-01

    ABSTRACT Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector. PMID:28594267

  8. Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

    OpenAIRE

    Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

    2012-01-01

    Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of ...

  9. Alemtuzumab-induced elimination of HIV-1-infected immune cells.

    Science.gov (United States)

    Ruxrungtham, Kiat; Sirivichayakul, Sunee; Buranapraditkun, Supranee; Krause, Werner

    2016-01-01

    Currently, there is no drug known that is able to eradicate either HIV or HIV-infected host cells. The effectiveness of all available treatments is based on the prevention of viral replication. We investigated whether the monoclonal, CD52 receptor-targeting antibody, alemtuzumab, which is currently approved for the treatment of multiple sclerosis, is able to eliminate HIV-infected immune cells. In blood samples from healthy donors and from HIV-1-infected subjects who were either treatment-naïve or resistant to HAART, we studied whether the CD52 expression on T cells and their subsets (CD3, CD4, CD8), B cells (CD19), dendritic cells (CD123) and monocytes (CD11c) is retained in HIV-1 infection and whether alemtuzumab is able to eradicate infected cells, using four-colour flow cytometry. We found that CD52 expression on immune cells is retained in HIV-1 infection regardless of CD4 cell count, viral load and treatment status, and is amenable to alemtuzumab-induced depletion. For the first time it could be shown in vitro that HIV-1-infected immune cells can be eliminated by using the monoclonal antibody alemtuzumab.

  10. Nipah virus infection and glycoprotein targeting in endothelial cells

    Directory of Open Access Journals (Sweden)

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  11. Stem Cell Transplant Patients and Fungal Infections

    Science.gov (United States)

    ... Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch Stem Cell Transplant Patients and Fungal Infections Recommend on Facebook ... Mold . Top of Page Preventing fungal infections in stem cell transplant patients Fungi are difficult to avoid because ...

  12. Establishment of human papillomavirus infection requires cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Dohun Pyeon

    2009-02-01

    Full Text Available Human papillomaviruses (HPVs are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these

  13. Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

    International Nuclear Information System (INIS)

    Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

    2004-01-01

    Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

  14. Immunity: Insect Immune Memory Goes Viral.

    Science.gov (United States)

    Ligoxygakis, Petros

    2017-11-20

    Adaptive memory in insect immunity has been controversial. In this issue, Andino and co-workers propose that acquisition of viral sequences in the host genome gives rise to anti-sense, anti-viral piRNAs. Such sequences can be regarded as both a genomic archive of past infections and as an armour of potential heritable memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Seasonal Abundance and Natural Inoculativity of Insect Vectors of Xylella fastidiosa in Oklahoma Tree Nurseries and Vineyards.

    Science.gov (United States)

    Overall, Lisa M; Rebek, Eric J

    2015-12-01

    Xylella fastidiosa is the causative agent of diseases of perennial plants including peach, plum, elm, oak, pecan, and grape. This bacterial pathogen is transmitted by xylem-feeding insects. In recent years, Pierce's disease of grape has been detected in 10 counties in central and northeastern Oklahoma, prompting further investigation of the disease epidemiology in this state. We surveyed vineyards and tree nurseries in Oklahoma for potential insect vectors to determine species composition, infectivity, and natural inoculativity of commonly captured insect vectors. Yellow sticky cards were used to sample insect fauna at each location. Insects were removed from sticky cards and screened for X. fastidiosa using immunocapture-PCR to determine their infectivity. A second objective was to test the natural inoculativity of insect vectors that are found in vineyards. Graphocephala versuta (Say), Graphocephala coccinea (Forster), Paraulacizes irrorata (F.), Oncometopia orbona (F.), Cuerna costalis (F.), and Entylia carinata Germar were collected from vineyards and taken back to the lab to determine their natural inoculativity. Immunocapture-PCR was used to test plant and insect samples for presence of X. fastidiosa. The three most frequently captured species from vineyards and tree nurseries were G. versuta, Clastoptera xanthocephala Germar, and O. orbona. Of those insects screened for X. fastidiosa, 2.4% tested positive for the bacterium. Field-collected G. versuta were inoculative to both ragweed and alfalfa. Following a 7-d inoculation access period, a higher percentage of alfalfa became infected than ragweed. Results from this study provide insight into the epidemiology of X. fastidiosa in Oklahoma. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Infection of endothelial cells by common human viruses.

    Science.gov (United States)

    Friedman, H M

    1989-01-01

    Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

  17. Early events associated with infection of Epstein-Barr virus infection of primary B-cells.

    Directory of Open Access Journals (Sweden)

    Sabyasachi Halder

    2009-09-01

    Full Text Available Epstein Barr virus (EBV is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology was used to introduce an expression cassette of green fluorescent protein (GFP by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

  18. The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

    Science.gov (United States)

    Han, Yingying; Xia, Hengchuan; Tang, Qi; Lü, Peng; Ma, Shangshang; Yang, Yanhua; Shao, Dandan; Ma, Quanbing; Chen, Keping

    2014-07-01

    The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

  19. Social immunity and the evolution of group living in insects.

    Science.gov (United States)

    Meunier, Joël

    2015-05-26

    The evolution of group living requires that individuals limit the inherent risks of parasite infection. To this end, group living insects have developed a unique capability of mounting collective anti-parasite defences, such as allogrooming and corpse removal from the nest. Over the last 20 years, this phenomenon (called social immunity) was mostly studied in eusocial insects, with results emphasizing its importance in derived social systems. However, the role of social immunity in the early evolution of group living remains unclear. Here, I investigate this topic by first presenting the definitions of social immunity and discussing their applications across social systems. I then provide an up-to-date appraisal of the collective and individual mechanisms of social immunity described in eusocial insects and show that they have counterparts in non-eusocial species and even solitary species. Finally, I review evidence demonstrating that the increased risks of parasite infection in group living species may both decrease and increase the level of personal immunity, and discuss how the expression of social immunity could drive these opposite effects. By highlighting similarities and differences of social immunity across social systems, this review emphasizes the potential importance of this phenomenon in the early evolution of the multiple forms of group living in insects. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  20. Human innate lymphoid cells (ILCs) in filarial infections.

    Science.gov (United States)

    Bonne-Année, S; Nutman, T B

    2018-02-01

    Filarial infections are characteristically chronic and can cause debilitating diseases governed by parasite-induced innate and adaptive immune responses. Filarial parasites traverse or establish niches in the skin (migrating infective larvae), in nonmucosal tissues (adult parasite niche) and in the blood or skin (circulating microfilariae) where they intersect with the host immune response. While several studies have demonstrated that filarial parasites and their antigens can modulate myeloid cells (monocyte, macrophage and dendritic cell subsets), T- and B-lymphocytes and skin resident cell populations, the role of innate lymphoid cells during filarial infections has only recently emerged. Despite the identification and characterization of innate lymphoid cells (ILCs) in murine helminth infections, little is actually known about the role of human ILCs during parasitic infections. The focus of this review will be to highlight the composition of ILCs in the skin, lymphatics and blood; where the host-parasite interaction is well-defined and to examine the role of ILCs during filarial infections. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  1. Entry into Midgut Epithelial Cells is a Key Step in the Selection of Genotypes in a Nucleopolyhedrovirus

    Institute of Scientific and Technical Information of China (English)

    Gabriel Clavijo; Trevor Williams; Delia Mu(n)oz; Miguel L(o)pez-Ferber; Primitivo Caballero

    2009-01-01

    An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.

  2. Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

    Science.gov (United States)

    Bernard-Stoecklin, Sibylle; Gommet, Céline; Corneau, Aurélien B.; Guenounou, Sabrina; Torres, Claire; Dejucq-Rainsford, Nathalie; Cosma, Antonio; Dereuddre-Bosquet, Nathalie; Le Grand, Roger

    2013-01-01

    The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4+ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4+ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure. PMID:24348253

  3. RNA Interference in Insect Vectors for Plant Viruses

    Directory of Open Access Journals (Sweden)

    Surapathrudu Kanakala

    2016-12-01

    Full Text Available Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

  4. Patterns of host adaptation in fly infecting Entomophthora species

    DEFF Research Database (Denmark)

    de Fine Licht, Henrik Hjarvard; Jensen, Annette Bruun; Eilenberg, Jørgen

    Insect pathogenic fungi (IPF) differ widely in their capability to infect different hosts. Some are generalists and will, given a sufficient number of infectious spores are present, infect almost any species of insect (e.g. Hypocrealean Metarhizium and Beauveria). Members of a different main IPF ...

  5. Infectious mononucleosis accompanied by clonal proliferation of EBV-infected cells and infection of CD8-positive cells.

    Science.gov (United States)

    Arai, Ayako; Yamaguchi, Takeshi; Komatsu, Honami; Imadome, Ken-Ichi; Kurata, Morito; Nagata, Kaoru; Miura, Osamu

    2014-01-01

    A 22-year-old male was admitted for a sustained fever of 2 months, lymphadenopathy, and liver dysfunction. Anti-VCA-IgM antibody was positive, with elevated Epstein-Barr virus (EBV)-DNA load in the peripheral blood. Liver biopsy revealed infiltration of CD8-positive and EBV-positive cells. Most peripheral blood mononuclear cells (PBMCs) were also positive for CD8, and showed detectable levels of EBV-DNA. Monoclonal proliferation of EBV-infected cells was detected in the PBMCs by Southern blotting for EBV-terminal repeat (EBV-TR). Although EBV-positive T-cell lymphoproliferative disease (EBV-T-LPD) was suspected, the symptoms spontaneously resolved within 12 months. Anti-VCA-IgM antibody and the clonal band of EBV-TR were negative 1 year after the onset, while anti-EBNA antibody was positive. The final diagnosis was thus confirmed as infectious mononucleosis (IM). Our results indicate that EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells may be temporally detected in IM. EBV-T-LPDs should be carefully excluded in such cases.

  6. Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

    Science.gov (United States)

    Ben Moussa, Issam Eddine; Mazzoni, Valerio; Valentini, Franco; Yaseen, Thaer; Lorusso, Donato; Speranza, Stefano; Digiaro, Michele; Varvaro, Leonardo; Krugner, Rodrigo; D'Onghia, Anna Maria

    2016-08-01

    A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Ultrastructural and pathogenesis of Monodon baculovirus in SPF shrimp, Litopenaeus vannamei imported to Iran

    OpenAIRE

    Bahari-Meimandi, S.A.; Afsharnasab, M.; Motallebi Moghanjoghi, A.A.; Azaritakami, G.; Sharifrohani, M.

    2014-01-01

    Viral pathogens are major causes of outbreaks in shrimp farms throughout the world. Monodon baculovirus has been known to be invasive in 85-100% of the shrimp hatcheries, in early or late stages of shrimp. Three-hundred and sixty juvenile of Litopenaeus vannamei with average (±SD) size of 7.99±0.54 g and 3600 post larvae 10-15 were prepared from Shrimp Research Station located in Helleh and 3 hatcheries from Bushehr Province, southern part of Iran, respectively. They were allocated to 9 glass...

  8. Susceptibility of different leukocyte cell types to Vaccinia virus infection

    Directory of Open Access Journals (Sweden)

    Sánchez-Puig Juana M

    2004-11-01

    Full Text Available Abstract Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP, and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.

  9. Effect of UV on DNA synthesis in UV-resistant insect cells

    International Nuclear Information System (INIS)

    Styer, S.C.; Meechan, P.J.; Griffiths, T.D.

    1987-01-01

    Insect cells are most resistant to killing by 254 nm ultraviolet light (UV) than mammalian cells. Because they have an active photolyase, it may be possible to generate a higher number of [6-4] PyC lesions per genome, allowing the possibility to distinguish between the effects of [5-6] pyrimidine lesions and the nonphotoreactable [6-4] lesions on DNA replication. IAL-PID2 cells, derived from imaginal wing discs of the Indian meal moth were exposed to UV followed by photoreactivating light (PR) or sham treatment and then analyzed by measuring the incorporation of [/sup 3/H]-thymidine into acid precipitable form. As expected, there was a fluence-dependent decrease in the amount of thymidine incorporated after exposure to UV. The response was similar to that observed in wild type CHO cells (AAS) except that the rate of decline was more rapid. When PR followed UV, there was less of a decline in thymidine incorporation and a more rapid recovery. However, thymidine incorporation did not return to control levels as rapidly as expected if [5-6] lesions were the only lesions involved in the disruption of DNA synthesis after exposure to UV

  10. Diversity and role of cave-dwelling hematophagous insects in pathogen transmission in the Afrotropical region.

    Science.gov (United States)

    Obame-Nkoghe, Judicaël; Leroy, Eric-Maurice; Paupy, Christophe

    2017-04-12

    The progressive anthropization of caves for food resources or economic purposes increases human exposure to pathogens that naturally infect cave-dwelling animals. The presence of wild or domestic animals in the immediate surroundings of caves also may contribute to increasing the risk of emergence of such pathogens. Some zoonotic pathogens are transmitted through direct contact, but many others require arthropod vectors, such as blood-feeding insects. In Africa, hematophagous insects often play a key role in the epidemiology of many pathogens; however, their ecology in cave habitats remains poorly known. During the last decades, several investigations carried out in Afrotropical caves suggested the medical and veterinary importance particularly of insect taxa of the Diptera order. Therefore, the role of some of these insects as vectors of pathogens that infect cave-dwelling vertebrates has been studied. The present review summarizes these findings, brings insights into the diversity of cave-dwelling hematophagous Diptera and their involvement in pathogen transmission, and finally discusses new challenges and future research directions.

  11. Dendritic cells during Epstein Barr virus infection

    Directory of Open Access Journals (Sweden)

    Christian eMunz

    2014-06-01

    Full Text Available Epstein Barr virus (EBV causes persistent infection in more than 90% of the human adult population and is associated with 2% of all tumors in humans. This -herpesvirus infects primarily human B and epithelial cells, but has been reported to be sensed by dendritic cells (DCs during primary infection. These activated DCs are thought to contribute to innate restriction of EBV infection and initiate EBV specific adaptive immune responses via cross-priming. The respective evidence and their potential importance for EBV specific vaccine development will be discussed in this review.

  12. Memory and Specificity in the Insect Immune System: Current Perspectives and Future Challenges

    Directory of Open Access Journals (Sweden)

    Dustin Cooper

    2017-05-01

    Full Text Available The immune response of a host to a pathogen is typically described as either innate or adaptive. The innate form of the immune response is conserved across all organisms, including insects. Previous and recent research has focused on the nature of the insect immune system and the results imply that the innate immune response of insects is more robust and specific than previously thought. Priming of the insect innate immune system involves the exposure of insects to dead or a sublethal dose of microbes in order to elicit an initial response. Comparing subsequent infections in primed insects to non-primed individuals indicates that the insect innate immune response may possess some of the qualities of an adaptive immune system. Although some studies demonstrate that the protective effects of priming are due to a “loitering” innate immune response, others have presented more convincing elements of adaptivity. While an immune mechanism capable of producing the same degree of recognition specificity as seen in vertebrates has yet to be discovered in insects, a few interesting cases have been identified and discussed.

  13. Characterization of a Bombyx mori nucleopolyhedrovirus with Bmvp80 disruption.

    Science.gov (United States)

    Tang, Xu-Dong; Xu, Yi-Peng; Yu, Lin-Lin; Lang, Guo-Jun; Tian, Cai-Hong; Zhao, Jin-Fang; Zhang, Chuan-Xi

    2008-12-01

    A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.

  14. HBV-Specific shRNA is Capable of Reducing the Formation of Hepatitis B Virus Covalently Closed Circular DNA, but has No Effect on Established Covalently Closed Circular DNA in vitro

    OpenAIRE

    Starkey, Jason L.; Chiari, Estelle F.; Isom, Harriet C.

    2009-01-01

    Hepatitis B virus (HBV) covalently closed circular DNA (CCC DNA) is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV expressing HepG2 cells at 10 days post-transduction ge...

  15. Evolution of specific immunity in shrimp - a vaccination perspective against white spot syndrome virus.

    Science.gov (United States)

    Syed Musthaq, Syed Khader; Kwang, Jimmy

    2014-10-01

    Invertebrates lack true adaptive immunity and it solely depends on the primitive immunity called innate immunity. However, various innate immune molecules and mechanisms are identified in shrimp that plays potential role against invading bacterial, fungal and viral pathogens. Perceiving the shrimp innate immune mechanisms will contribute in developing effective vaccine strategies against major shrimp pathogens. Hence this review intends to explore the innate immune molecules of shrimp with suitable experimental evidences together with the evolution of "specific immune priming" of invertebrates. In addition, we have emphasized on the development of an effective vaccine strategy against major shrimp pathogen, white spot syndrome virus (WSSV). The baculovirus displayed rVP28 (Bac-VP28), a major envelope protein of WSSV was utilized to study its vaccine efficacy by oral route. A significant advantage of this baculovirus expression cassette is the use of WSSV-immediate early 1 (ie1) promoter that derived the abundant expression of rVP28 protein at the early stage of the infection in insect cell. The orally vaccinated shrimp with Bac-VP28 transduced successfully in the shrimp cells as well as provided highest survival rate. In support to our vaccine efficacy we analysed Pattern Recognition Proteins (PRPs) β-1,3 glucan lipopolysaccharides (LGBP) and STAT gene profiles in the experimental shrimp. Indeed, the vaccination of shrimp with Bac-VP28 demonstrated some degree of specificity with enhanced survival rate when compared to control vaccination with Bac-wt. Hence it is presumed that the concept of "specific immune priming" in relevant to shrimp immunity is possible but may not be common to all shrimp pathogens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Reprint of "evolution of specific immunity in shrimp - a vaccination perspective against white spot syndrome virus".

    Science.gov (United States)

    Syed Musthaq, Syed Khader; Kwang, Jimmy

    2015-02-01

    Invertebrates lack true adaptive immunity and it solely depends on the primitive immunity called innate immunity. However, various innate immune molecules and mechanisms are identified in shrimp that plays potential role against invading bacterial, fungal and viral pathogens. Perceiving the shrimp innate immune mechanisms will contribute in developing effective vaccine strategies against major shrimp pathogens. Hence this review intends to explore the innate immune molecules of shrimp with suitable experimental evidences together with the evolution of "specific immune priming" of invertebrates. In addition, we have emphasized on the development of an effective vaccine strategy against major shrimp pathogen, white spot syndrome virus (WSSV). The baculovirus displayed rVP28 (Bac-VP28), a major envelope protein of WSSV was utilized to study its vaccine efficacy by oral route. A significant advantage of this baculovirus expression cassette is the use of WSSV-immediate early 1 (ie1) promoter that derived the abundant expression of rVP28 protein at the early stage of the infection in insect cell. The orally vaccinated shrimp with Bac-VP28 transduced successfully in the shrimp cells as well as provided highest survival rate. In support to our vaccine efficacy we analysed Pattern Recognition Proteins (PRPs) β-1,3 glucan lipopolysaccharides (LGBP) and STAT gene profiles in the experimental shrimp. Indeed, the vaccination of shrimp with Bac-VP28 demonstrated some degree of specificity with enhanced survival rate when compared to control vaccination with Bac-wt. Hence it is presumed that the concept of "specific immune priming" in relevant to shrimp immunity is possible but may not be common to all shrimp pathogens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Immune regulation in Chandipura virus infection: characterization of CD4+ T regulatory cells from infected mice

    Directory of Open Access Journals (Sweden)

    Shahir Prajakta

    2011-05-01

    Full Text Available Abstract Back ground Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several mechanisms like generation of regulatory cells, activation induced cell death (ACID etc were indicated to control the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice. Method In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+ T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were studied by flowcytometer. Results The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3 antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis and light microscopy revealed that CD4 negative cells were of different size and shape (atypical compared to the normal lymphocytes. Greater percentage of

  18. MAIT cells are activated in acute Dengue virus infection and after in vitro Zika virus infection.

    Directory of Open Access Journals (Sweden)

    Dominic Paquin-Proulx

    2018-01-01

    Full Text Available Dengue virus (DENV and Zika virus (ZIKV are members of the Flaviviridae and are predominantly transmitted via mosquito bites. Both viruses are responsible for a growing number of infections in tropical and subtropical regions. DENV infection can cause lethargy with severe morbidity and dengue shock syndrome leading to death in some cases. ZIKV is now linked with Guillain-Barré syndrome and fetal malformations including microcephaly and developmental disorders (congenital Zika syndrome. The protective and pathogenic roles played by the immune response in these infections is unknown. Mucosal-associated invariant T (MAIT cells are a population of innate T cells with potent anti-bacterial activity. MAIT cells have also been postulated to play a role in the immune response to viral infections. In this study, we evaluated MAIT cell frequency, phenotype, and function in samples from subjects with acute and convalescent DENV infection. We found that in acute DENV infection, MAIT cells had elevated co-expression of the activation markers CD38 and HLA-DR and had a poor IFNγ response following bacterial stimulation. Furthermore, we found that MAIT cells can produce IFNγ in response to in vitro infection with ZIKV. This MAIT cell response was independent of MR1, but dependent on IL-12 and IL-18. Our results suggest that MAIT cells may play an important role in the immune response to Flavivirus infections.

  19. Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells.

    Science.gov (United States)

    Mayer, Bryan T; Krantz, Elizabeth M; Swan, David; Ferrenberg, James; Simmons, Karen; Selke, Stacy; Huang, Meei-Li; Casper, Corey; Corey, Lawrence; Wald, Anna; Schiffer, Joshua T; Gantt, Soren

    2017-06-15

    Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (<1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine. IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We

  20. Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

    Science.gov (United States)

    Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

    2015-06-01

    Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

  1. Colored and white sectors of petunia flowers display differential resistance to insect herbivores

    Science.gov (United States)

    Insect herbivory of crops increases the probability of fungal infection in damaged tissues. Mycotoxins produced by some fungi are harmful to livestock and humans. Increasing plant resistance lowers the levels of fungal infection and mycotoxin levels. The Bt toxin successfully kills only a fractio...

  2. Retroviral infection of non-dividing cells: Old and new perspectives

    International Nuclear Information System (INIS)

    Yamashita, Masahiro; Emerman, Michael

    2006-01-01

    The dependence of retroviral replication on cell proliferation was described as early as 1958, although different classes of retroviruses are able to infect non-dividing cells with different efficiencies. For example, the human immunodeficiency virus (HIV) and other lentiviruses infect most non-dividing cells nearly as well as dividing cells, while the gammaretroviruses such as the murine leukemia virus (MLV) cannot infect non-dividing cells, and other retroviruses have intermediate phenotypes. One exception to the ability of HIV to infect non-dividing cells involves resting CD4+ T cells in vitro where there are multiple restrictions. However, recent data show that there is massive infection of non-activated CD4+ T cell during acute infection which suggests that the situation is different in vivo. Finally, much work trying to explain the difference between HIV and MLV in non-dividing cells has focused on describing the ability of HIV to enter the nucleus during interphase. However, we suggest that events in the viral lifecycle other than nuclear import may be more important in determining the ability of a given retrovirus to infect non-dividing cells

  3. T Follicular Helper Cells and B Cell Dysfunction in Aging and HIV-1 Infection.

    Science.gov (United States)

    Pallikkuth, Suresh; de Armas, Lesley; Rinaldi, Stefano; Pahwa, Savita

    2017-01-01

    T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh-B cell interactions.

  4. Detrimental and Neutral Effects of a Wild Grass-Fungal Endophyte Symbiotum on Insect Preference and Performance

    OpenAIRE

    Clement, Stephen L.; Hu, Jinguo; Stewart, Alan V.; Wang, Bingrui; Elberson, Leslie R.

    2011-01-01

    Seed-borne Epichloë/Neotyphodium Glenn, Bacon, Hanlin (Ascomycota: Hypocreales: Clavicipitaceae) fungal endophytes in temperate grasses can provide protection against insect attack with the degree of host resistance related to the grass—endophyte symbiotum and the insect species involved in an interaction. Few experimental studies with wild grass—endophyte symbiota, compared to endophyte-infected agricultural grasses, have tested for anti-insect benefits, let alone for resistance against more...

  5. Localization and dynamics of Wolbachia infection in Asian citrus psyllid Diaphorina citri, the insect vector of the causal pathogens of Huanglongbing.

    Science.gov (United States)

    Ren, Su-Li; Li, Yi-Han; Ou, Da; Guo, Yan-Jun; Qureshi, Jawwad A; Stansly, Philip A; Qiu, Bao-Li

    2018-03-23

    Wolbachia is a group of intracellular bacteria that infect a wide range of arthropods including the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. This insect is the vector of Candidatus Liberibacter asiaticus (CLas), the causal pathogen of Huanglongbing or citrus greening disease. Here, we investigated the localization pattern and infection dynamics of Wolbachia in different developmental stages of ACP. Results revealed that all developmental stages of ACP including egg, 1st-5th instar nymphs, and adults of both gender were infected with Wolbachia. FISH visualization of an ACP egg showed that Wolbachia moved from the egg stalk of newly laid eggs to a randomly distributed pattern throughout the egg prior to hatching. The infection rate varied between nymphal instars. The titers of Wolbachia in fourth and fifth instar nymphs were significantly higher than those in the first and second instar nymphs. Wolbachia were scattered in all nymphal stages, but with highest intensity in the U-shaped bacteriome located in the abdomen of the nymph. Wolbachia was confined to two symmetrical organizations in the abdomen of newly emerged female and male adults. The potential mechanisms of Wolbachia infection dynamics are discussed. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. Recombinant culicoides obsoletus complex allergens stimulate antigen-specific T cells on insect bite hypersensitive Shetland ponies in vitro

    NARCIS (Netherlands)

    Meulenbroeks, C.; Meide, van der N.M.A.; Willemse, T.; Rutten, V.; Tijhaar, E.J.

    2015-01-01

    Background Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. Hypothesis/Objectives To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of

  7. Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

    Directory of Open Access Journals (Sweden)

    Manuela Fogli

    2008-07-01

    Full Text Available Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7. This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I molecules, HIV-1-infected p24(pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs and with the high frequency of the anergic CD56(neg/CD16(pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos blasts derived from primary T cells.

  8. Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Stephen H-F Macdonald

    Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

  9. Transcriptomic response of the insect vector, Peregrinus maidis, to Maize mosaic rhabdovirus and identification of conserved responses to propagative viruses in hopper vectors.

    Science.gov (United States)

    Martin, Kathleen M; Barandoc-Alviar, Karen; Schneweis, Derek J; Stewart, Catherine L; Rotenberg, Dorith; Whitfield, Anna E

    2017-09-01

    Maize mosaic virus (MMV) is a plant-pathogenic rhabdovirus that is transmitted by the corn planthopper, Peregrinus maidis, in a propagative manner. P. maidis supports long-term MMV infections with no negative effects on insect performance. To elucidate whole-body transcriptome responses to virus infection, RNA-Seq was used to examine differential gene expression of virus-infected adult insects, and libraries were prepared from replicated groups of virus-exposed insects and non-exposed insects. From the 68,003 de novo-assembled transcripts, 144 were differentially-expressed (DE) during viral infection with comparable numbers up- and down-regulated. DE transcripts with similarity to genes associated with transposable elements (i.e., RNA-directed DNA polymerases) were enriched and may represent a mechanisim for modulating virus infection. Comparison of the P. maidis DE transcripts to published propagative virus-responsive transcript databases for two other hopper vectors revealed that 16% of the DE transcripts were shared across the three systems and may represent conserved responses to propagative viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

    Directory of Open Access Journals (Sweden)

    Shrilakshmi Hegde

    Full Text Available Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

  11. N-glycan sialylation in a silkworm-baculovirus expression system.

    Science.gov (United States)

    Suganuma, Masatoshi; Nomura, Tsuyoshi; Higa, Yukiko; Kataoka, Yukiko; Funaguma, Shunsuke; Okazaki, Hironobu; Suzuki, Takeo; Fujiyama, Kazuhito; Sezutsu, Hideki; Tatematsu, Ken-Ichiro; Tamura, Toshiki

    2018-02-09

    A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Riboflavin Provisioning Underlies Wolbachia's Fitness Contribution to Its Insect Host.

    Science.gov (United States)

    Moriyama, Minoru; Nikoh, Naruo; Hosokawa, Takahiro; Fukatsu, Takema

    2015-11-10

    Endosymbiotic bacteria of the genus Wolbachia represent the most successful symbiotic bacteria in the terrestrial ecosystem. The success of Wolbachia has been ascribed to its remarkable phenotypic effects on host reproduction, such as cytoplasmic incompatibility, whereby maternally inherited bacteria can spread in their host populations at the expense of their host's fitness. Meanwhile, recent theoretical as well as empirical studies have unveiled that weak and/or conditional positive fitness effects may significantly facilitate invasion and spread of Wolbachia infections in host populations. Here, we report a previously unrecognized nutritional aspect, the provision of riboflavin (vitamin B2), that potentially underpins the Wolbachia-mediated fitness benefit to insect hosts. A comparative genomic survey for synthetic capability of B vitamins revealed that only the synthesis pathway for riboflavin is highly conserved among diverse insect-associated Wolbachia strains, while the synthesis pathways for other B vitamins were either incomplete or absent. Molecular phylogenetic and genomic analyses of riboflavin synthesis genes from diverse Wolbachia strains revealed that, in general, their phylogenetic relationships are concordant with Wolbachia's genomic phylogeny, suggesting that the riboflavin synthesis genes have been stably maintained in the course of Wolbachia evolution. In rearing experiments with bedbugs (Cimex lectularius) on blood meals in which B vitamin contents were manipulated, we demonstrated that Wolbachia's riboflavin provisioning significantly contributes to growth, survival, and reproduction of the insect host. These results provide a physiological basis upon which Wolbachia-mediated positive fitness consequences are manifested and shed new light on the ecological and evolutionary relevance of Wolbachia infections. Conventionally, Wolbachia has been regarded as a parasitic bacterial endosymbiont that manipulates the host insect's reproduction in a

  13. Sterile insect technique and radiation in insect control

    International Nuclear Information System (INIS)

    1982-01-01

    Out of 39 papers and 6 summaries of the poster presentations published in this proceeding series, 23 respectively fall within the INIS subject scope. Four main topics were covered: a review of the sterile insect technique against various insect pests; its application to tsetse flies in eradication programmes; quality control of mass-reared insects for release; and the development of genetic approaches to insect mass rearing and control. Other topics emphasized integrated pest management, computer models and radioisotope labelling

  14. Quantitative comparison of HTLV-1 and HIV-1 cell-to-cell infection with new replication dependent vectors.

    Directory of Open Access Journals (Sweden)

    Dmitriy Mazurov

    2010-02-01

    Full Text Available We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

  15. Development and applications of VSV vectors based on cell tropism

    Directory of Open Access Journals (Sweden)

    Hideki eTani

    2012-01-01

    Full Text Available Viral vectors have been available in various fields such as medical and biological research or gene therapy applications. Targeting vectors pseudotyped with distinct viral envelope proteins that influence cell tropism and transfection efficiency is a useful tool not only for examining entry mechanisms or cell tropisms but also for vaccine vector development. Vesicular stomatitis virus (VSV is an excellent candidate for development as a pseudotype vector. A recombinant VSV lacking its own envelope (G gene has been used to produce a pseudotype or recombinant VSV possessing the envelope proteins of heterologous viruses. These viruses possess a reporter gene instead of a VSV G gene in their genome, and therefore it is easy to evaluate their infectivity in the study of viral entry, including identification of viral receptors. Furthermore, advantage can be taken of a property of the pseudotype VSV, which is competence for single-round infection, in handling many different viruses that are either difficult to amplify in cultured cells or animals or that require specialized containment facilities. Here we describe procedures for producing pseudotype or recombinant VSVs and a few of the more prominent examples from among envelope viruses, such as hepatitis C virus, Japanese encephalitis virus, baculovirus, and hemorrhagic fever viruses.

  16. Anatomy of adult Megaphragma (Hymenoptera: Trichogrammatidae, one of the smallest insects, and new insight into insect miniaturization.

    Directory of Open Access Journals (Sweden)

    Alexey A Polilov

    Full Text Available The body size, especially in cases of extreme reduction, is an important characteristic that strongly determines the morphology, physiology, and biology of animals. Miniaturization is a widespread trend in animal evolution and one of the principal directions of evolution in insects. Miniaturization-related features of insect morphology have been subject to intensive studies during the last few years, but the structure of the smallest insects remains insufficiently known. It is especially important to study hymenopterans of the genus Megaphragma, which include the smallest flying insects and a species in which an almost anucleate nervous system was recently discovered. This article is the first detailed study of the external and internal morphology of adults of Megaphragma mymaripenne and M. amalphitanum using histological methods, 3D computer modeling and other techniques. It is shown that in spite of the extremely small size the organization of Megaphragma retains a considerkable level of structural complexity. On the other hand, miniaturization leads to re-organizations of several organ systems. Unique structural features related to miniaturization have been found in both species: lysis of cell bodies and nuclei of neurons at late stages of pupal development, absence of the heart, and considerable reductions in the set of muscles. Comparative analysis of structure in the smallest insects representing different taxa has revealed common features of the evolutionary process of miniaturization in insects.

  17. Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

    Science.gov (United States)

    Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

    2018-01-01

    SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484

  18. Regulation of NKT Cell Localization in Homeostasis and Infection

    Science.gov (United States)

    Slauenwhite, Drew; Johnston, Brent

    2015-01-01

    Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection. PMID:26074921

  19. Regulation of NKT Cell Localization in Homeostasis and Infection.

    Science.gov (United States)

    Slauenwhite, Drew; Johnston, Brent

    2015-01-01

    Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection.

  20. Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

    Science.gov (United States)

    Cha, Hefei; Qin, Wenjuan; Yang, Quan; Xie, Hongyan; Qu, Jiale; Wang, Mei; Chen, Daixiong; Wang, Fang; Dong, Nuo; Chen, Longhua; Huang, Jun

    2017-02-01

    Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P cell number of NKT cells were decreased compared to those of normal mice (P NKT cells was increased after infection (P NKT cells (P cells (P NKT cells significantly increased (P NKT cells (P NKT cell activation during S. japonicum infection.

  1. Aromatic/heterocyclic amino acids and the simulated sunlight-ultraviolet inactivation of the Heliothis/Helicoverpa baculovirus

    International Nuclear Information System (INIS)

    Ignoffo, C.M.; Garcia, C.

    1995-01-01

    Tryptophan, of five aromatic/heterocyclic amino acids (tyrosine, phenylalanine, proline, histidine) provided significant protection of the Heliothis baculovirus (HzSNPV) from inactivation by simulated ultraviolet (SUV). Fifty percent of SUV protection of HzSNPV with tryptophan or tyrosine was obtained at 0.03 mg/ml and 0.5 mg/ml, respectively. Rates as high as 100.0 mg/ml of phenylalanine, histidine, or proline provided <50% protection. The extent of tryptophan protection was correlated with its absorption in the sunlight UV-B spectra. 16 refs., 2 tabs

  2. Detrimental and neutral effects of a wild grass-fungal endophyte symbiotum on insect preference and performance.

    Science.gov (United States)

    Clement, Stephen L; Hu, Jinguo; Stewart, Alan V; Wang, Bingrui; Elberson, Leslie R

    2011-01-01

    Seed-borne Epichloë/Neotyphodium Glenn, Bacon, Hanlin (Ascomycota: Hypocreales: Clavicipitaceae) fungal endophytes in temperate grasses can provide protection against insect attack with the degree of host resistance related to the grass-endophyte symbiotum and the insect species involved in an interaction. Few experimental studies with wild grass-endophyte symbiota, compared to endophyte-infected agricultural grasses, have tested for anti-insect benefits, let alone for resistance against more than one insect species. This study quantified the preference and performance of the bird cherry oat-aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae) and the cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae), two important pests of forage and cereal grasses, on Neotyphodium-infected (E+) and uninfected (E-) plants of the wild grass Alpine timothy, Phleum alpinum L. (Poales: Poaceae). The experiments tested for both constitutive and wound-induced resistance in E+ plants to characterize possible plasticity of defense responses by a wild E+ grass. The aphid, R. padi preferred E- over E+ test plants in choice experiments and E+ undamaged test plants constitutively expressed antibiosis resistance to this aphid by suppressing population growth. Prior damage of E+ test plants did not induce higher levels of resistance to R. padi. By contrast, the beetle, O. melanopus showed no preference for E+ or E- test plants and endophyte infection did not adversely affect the survival and development of larvae. These results extend the phenomenon of variable effects of E+ wild grasses on the preference and performance of phytophagous insects. The wild grass- Neotyphodium symbiotum in this study broadens the number of wild E+ grasses available for expanded explorations into the effects of endophyte metabolites on insect herbivory.

  3. CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.

    Science.gov (United States)

    Taning, Clauvis Nji Tizi; Van Eynde, Benigna; Yu, Na; Ma, Sanyuan; Smagghe, Guy

    2017-04-01

    Discovered as a bacterial adaptive immune system, CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR associated) is being developed as an attractive tool in genome editing. Due to its high specificity and applicability, CRISPR/Cas9-mediated gene editing has been employed in a multitude of organisms and cells, including insects, for not only fundamental research such as gene function studies, but also applied research such as modification of organisms of economic importance. Despite the rapid increase in the use of CRISPR in insect genome editing, results still differ from each study, principally due to existing differences in experimental parameters, such as the Cas9 and guide RNA form, the delivery method, the target gene and off-target effects. Here, we review current reports on the successes of CRISPR/Cas9 applications in diverse insects and insect cells. We furthermore summarize several best practices to give a useful checklist of CRISPR/Cas9 experimental setup in insects for beginners. Lastly, we discuss the biosafety concerns related to the release of CRISPR/Cas9-edited insects into the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. NK cell-like behavior of Valpha14i NK T cells during MCMV infection.

    Directory of Open Access Journals (Sweden)

    Johnna D Wesley

    2008-07-01

    Full Text Available Immunity to the murine cytomegalovirus (MCMV is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Valpha14 invariant natural killer T cell response to MCMV has not been examined. We found that Valpha14i NK T cells become activated and produce significant levels of IFN-gamma, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Valpha14i NK T cells into MCMV-infected CD1d(-/- mice demonstrate that CD1d is dispensable for Valpha14i NK T cell activation. In contrast, both IFN-alpha/beta and IL-12 are required for optimal activation. Valpha14i NK T cell-derived IFN-gamma is partially dependent on IFN-alpha/beta but highly dependent on IL-12. Valpha14i NK T cells contribute to the immune response to MCMV and amplify NK cell-derived IFN-gamma. Importantly, mortality is increased in CD1d(-/- mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Valpha14i NK T cells that act as effector T cells during bacterial infection, but have NK cell-like behavior during the innate immune response to MCMV infection.

  5. Effects of interferon on cultured cells persistently infected with viruses

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M

    1986-01-01

    The role of interferon (IFN) in viral persistence at the cellular level was investigated. Two types of persistent infections were chosen. The first type was cell lines which contained hepatitis B virus (HBV) DNA (PLC/PRF/5 and Hep 3B cells) uninfected control hepatoma cells, (Mahlavu, HA22T and Hep G2 cells) or simian virus 40 (SV40) DNA (C2, C6, C11 cells) and control uninfected (CV-1 cells). In the second type of infection Vero cells persistently infected with SSPE or Sendai virus were used. The aim of this work was to determine what effect IFN had in these infections in terms of its antiviral and antiproliferative effects; which of the two major IFN-induced pathways, E enzyme or protein kinase were induced; whether there were any differences in sensitivity to IFN between the DNA and RNA virus persistent infections. The anti-viral effect of IFN was examined by its ability to inhibit Sindbis virus replication using a radioimmunoassay system. The antiproliferative effect of IFN was determined by cell counting and /sup 3/H-thymidine incorporation. The activation of the ribonuclease F, determined by the inhibition of /sup 3/H-leucine incorporation after introduction of 2-5 actin into the cells, was variable, being activated in all cell lines with the exception of the PLC/PRF/5, Hep 3B and Hep G2 cells. Major differences between the two DNA persistent infections and the two RNA persistent infections were found. No correlation was found between the presence of HBV or SV40 persistent infections and the sensitivity of the cell lines to IFN. Both the SSPE and Sendai virus persistent infections were resistant to the antiviral and antiproliferative effect of IFN.

  6. Defective Natural Killer cell antiviral capacity in paediatric HBV infection

    DEFF Research Database (Denmark)

    Heiberg, Ida Louise; Laura J., Pallett; Winther, Thilde Nordmann

    2015-01-01

    Natural Killer (NK) cells exhibit dysregulated effector function in adult chronic HBV infection (CHB), which may contribute to virus persistence. The role of NK cells in children infected perinatally with HBV is less studied. Access to a unique cohort enabled the cross-sectional evaluation of NK...... cell frequency, phenotype and function in HBV-infected children relative to uninfected children. We observed a selective defect in NK cell IFN-γ production, with conserved cytolytic function, mirroring the functional dichotomy observed in adult infection. Reduced expression of NKp30 on NK cells...

  7. Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests.

    Science.gov (United States)

    Gillet, François-Xavier; Garcia, Rayssa A; Macedo, Leonardo L P; Albuquerque, Erika V S; Silva, Maria C M; Grossi-de-Sa, Maria F

    2017-01-01

    Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil ( Anthonomus grandis ), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

  8. Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests

    Directory of Open Access Journals (Sweden)

    François-Xavier Gillet

    2017-04-01

    Full Text Available Genetically modified (GM crops producing double-stranded RNAs (dsRNAs are being investigated largely as an RNA interference (RNAi-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis, we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain combined with dsRNA forms a ribonucleoprotein particle (RNP that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

  9. Invariant NKT cells: regulation and function during viral infection.

    Directory of Open Access Journals (Sweden)

    Jennifer A Juno

    Full Text Available Natural killer T cells (NKT cells represent a subset of T lymphocytes that express natural killer (NK cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT, express a highly restricted T cell receptor (TCR and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.

  10. Beneficial Insects and Insect Pollinators on Milkweed in South Georgia

    Science.gov (United States)

    Insect pollinators are essential for the reproduction of more than two-thirds of the world’s crops, and beneficial insects play an important role in managing pest insects in agricultural farmscapes. These insects depend on nectar for their survival in these farmscapes. The flowers of tropical milkwe...

  11. Stinging insect allergy: state of the art 2015.

    Science.gov (United States)

    Tankersley, Michael S; Ledford, Dennis K

    2015-01-01

    Stinging insect allergy is responsible for more than 10% of all cases of anaphylaxis. The potential culprit insects are diverse and vary with geography. The incidence of insect allergy is declining in some areas and increasing in others, possibly due to effects of climate change, introduction of species into new areas, outdoor recreational activities, and movement of human populations that brings insects into contact with a greater number of people. Flying Hymenoptera and imported fire ant stings are responsible for the majority of patients evaluated for insect anaphylaxis. The most efficient means of identifying allergy to insects is skin testing although falsely positive and negative results occur. The limitations of testing coupled with the natural temporal variability of allergic sensitivity complicate the interpretation of test results. The clinical history is of paramount importance to be certain that the test results are relevant; therefore, screening or testing before a history of a sting reaction is not advisable. Mast cell disorders are associated with severe anaphylaxis from insect stings and should be considered in affected subjects. Insect immunotherapy, using venoms for most insects and whole-body extracts for imported fire ants, is proven effective in reducing the likelihood of anaphylaxis due to subsequent stings from 40%-60% to less than 5%. Future clinical application of component testing or in vitro cellular tests, such as the basophil activation test, may improve optimal choices for immunotherapy. Published by Elsevier Inc.

  12. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

    Directory of Open Access Journals (Sweden)

    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  13. Infection of a mammal by monogenetic insect trypanosomatids (Kinetoplastida, trypanosomatidae

    Directory of Open Access Journals (Sweden)

    Ana M. Jansen

    1988-09-01

    Full Text Available Monogenetic insect trypanosomatids of the genera Crithidia, Leptomonas and Herpetomonas, multiplied as in axenic cultures, for many months, in the lumen of the scent glands of the opossum Didelphis marsupialis. Specific antibodies were detected in the serum of the animals but there was no evidence of invasion of their tissues by the parasites.

  14. P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    2013-02-01

    Full Text Available Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress-responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

  15. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

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    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  16. Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells.

    Science.gov (United States)

    Katri, Patricia; Ruan, Shigui

    2004-11-01

    Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.

  17. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

    Science.gov (United States)

    Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H; Michel, Jennifer Carlisle; Claxton, Derek P; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K Christopher; Gouaux, Eric

    2014-11-01

    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

  18. Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

    DEFF Research Database (Denmark)

    Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

    2007-01-01

    Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

  19. Entomopathogenic Fungi Associated with Exotic Invasive Insect Pests in Northeastern Forests of the USA

    Science.gov (United States)

    Gouli, Vladimir; Gouli, Svetlana; Marcelino, José A. P.; Skinner, Margaret; Parker, Bruce L.

    2013-01-01

    Mycopathogens of economically important exotic invasive insects in forests of northeastern USA have been the subject of research at the Entomology Research Laboratory, University of Vermont, for the last 20 years. Elongate hemlock scale, European fruit lecanium, hemlock woolly adelgid and pear thrips were analyzed for the presence of mycopathogens, in order to consider the potential for managing these pests with biological control. Fungal cultures isolated from insects with signs of fungal infection were identified based on morphological characters and DNA profiling. Mycopathogens recovered from infected insects were subdivided into three groups, i.e., specialized entomopathogenic; facultative entomopathogens; ubiquitous opportunistic contaminants. Epizootics were caused by fungi in the specialized group with the exception of M. microspora, P. marquandii and I. farinosa. Inoculation of insects in laboratory and field conditions with B. bassiana, L. muscarium and Myriangium sp. caused insect mortality of 45 to 95%. Although pest populations in the field seemed severely compromised after treatment, the remnant populations re-established themselves after the winter. Although capable of inducing high mortality, a single localized aerial application of a soil-dwelling fungus does not maintain long-time suppression of pests. However, it can halt their range expansion and maintain populations below the economic threshold level without the use of expensive insecticides which have a negative impact on the environment. PMID:26462527

  20. Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

    Science.gov (United States)

    Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

    2015-01-01

    Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high throughput proteomic assays, we have previously identified Bruton’s tyrosine kinase (BTK) as a host protein that was uniquely up-regulated in the plasma membrane of HIV-1 infected T-cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant up-regulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells, however new BTK protein complexes were identified and distributed in both high molecular weight (~600 kDa) and a small molecular weight complex (~60–120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1 infected cells using siRNA resulted in selective death of infected, but not uninfected, cells. Using BTK specific antibody and small molecule inhibitors including LFM-A13 and a FDA approved compound, Ibrutinib (PCI – 32765), we have found that HIV-1 infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1 infected cells are sensitive to treatments targeting BTK expressed in infected cells. PMID:25672887

  1. The homeostasis of Plasmodium falciparum-infected red blood cells.

    Directory of Open Access Journals (Sweden)

    Jakob M A Mauritz

    2009-04-01

    Full Text Available The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis. However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

  2. T-cell-dependent control of acute Giardia lamblia infections in mice.

    Science.gov (United States)

    Singer, S M; Nash, T E

    2000-01-01

    We have studied immune mechanisms responsible for control of acute Giardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice with G. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to control G. lamblia infections, as has been shown previously for G. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either alphabeta- or gammadelta-T-cell receptor (TCR)-expressing T cells, we show that the alphabeta-TCR-expressing T cells are required to control parasites but that the gammadelta-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection from G. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

  3. Natural Killer Cell Function and Dysfunction in Hepatitis C Virus Infection

    Directory of Open Access Journals (Sweden)

    Kayla A. Holder

    2014-01-01

    Full Text Available Viruses must continually adapt against dynamic innate and adaptive responses of the host immune system to establish chronic infection. Only a small minority (~20% of those exposed to hepatitis C virus (HCV spontaneously clear infection, leaving approximately 200 million people worldwide chronically infected with HCV. A number of recent research studies suggest that establishment and maintenance of chronic HCV infection involve natural killer (NK cell dysfunction. This relationship is illustrated in vitro by disruption of typical NK cell responses including both cell-mediated cytotoxicity and cytokine production. Expression of a number of activating NK cell receptors in vivo is also affected in chronic HCV infection. Thus, direct in vivo and in vitro evidence of compromised NK function in chronic HCV infection in conjunction with significant epidemiological associations between the outcome of HCV infection and certain combinations of NK cell regulatory receptor and class I human histocompatibility linked antigen (HLA genotypes indicate that NK cells are important in the immune response against HCV infection. In this review, we highlight evidence suggesting that selective impairment of NK cell activity is related to establishment of chronic HCV infection.

  4. Electron Microscopy of Ebola Virus-Infected Cells.

    Science.gov (United States)

    Noda, Takeshi

    2017-01-01

    Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

  5. Human neuronal cell protein responses to Nipah virus infection

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    Hassan Sharifah

    2007-06-01

    Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

  6. The evolutionary development of plant-feeding insects and their nutritional endosymbionts.

    Science.gov (United States)

    Skidmore, Isabel H; Hansen, Allison K

    2017-12-01

    Herbivorous insects have evolved diverse mechanisms enabling them to feed on plants with suboptimal nutrient availability. Low nutrient availability negatively impacts insect herbivore development and fitness. To overcome this obstacle numerous insect lineages have evolved intimate associations with nutritional endosymbionts. This is especially true for insects that specialize on nitrogen-poor substrates, as these insects are highly dependent on intracellular symbionts to provide nitrogen lacking in their insect host's diet. Emerging evidence in these systems suggest that the symbiont's and/or the insect's biosynthetic pathways are dynamically regulated throughout the insect's development to potentially cope with the insect's changing nutritional demands. In this review, we evaluate the evolutionary development of symbiotic insect cells (bacteriocytes) by comparing and contrasting genes and mechanisms involved in maintaining and regulating the nutritional symbiosis throughout insect development in a diversity of insect herbivore-endosymbiont associations. With new advances in genome sequencing and functional genomics, we evaluate to what extent nutritional symbioses are shaped by (i) the regulation of symbiont titer, (ii) the regulation of insect symbiosis genes, and (iii) the regulation of symbiont genes. We discuss how important these mechanisms are for the biosynthesis of essential amino acids and vitamins across insect life stages in divergent insect-symbiont systems. We conclude by suggesting future directions of research to further elucidate the evolutionary development of bacteriocytes and the impact of these nutritional symbioses on insect-plant interactions. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  7. Edible Insects

    NARCIS (Netherlands)

    Huis, van A.; Dunkel, F.V.

    2016-01-01

    The interest in insects as human food in the Western world is increasingly considered as a viable alternative to other protein sources. In tropical countries it is common practice and about 2000 insect species are eaten. Insects emit low levels of greenhouse gases, need little water, and require

  8. Adrenaline-induced mobilization of T cells in HIV-infected patients

    DEFF Research Database (Denmark)

    Søndergaard, S R; Cozzi-Lepri, A; Ullum, H

    2000-01-01

    The present study aimed to investigate lymphocyte mobilization from peripheral cell reservoirs in HIV-infected patients. Nine HIV-infected patients on stable highly active anti-retroviral therapy (HAART), eight treatment-naive HIV-infected patients and eight HIV- controls received a 1-h adrenaline...... infusion. The adrenaline infusion induced a three-fold increase in the concentration of lymphocytes in all three groups. All HIV-infected patients mobilized significantly higher numbers of CD8+ cells but less CD4+ cells. All subjects mobilized CD45RA+CD62L+ and CD8+CD28+ cells to a lesser extent than CD45......RO+CD45RA- and CD8+CD28-cells. Furthermore, high numbers of CD8+CD38+ cells were mobilized only in the HIV-infected patients. It was therefore predominantly T cells with an activated phenotype which were mobilized after adrenaline stimulation. It is concluded that the HIV-associated immune defect...

  9. Insects: A nutritional alternative

    Science.gov (United States)

    Dufour, P. A.

    1981-01-01

    Insects are considered as potential food sources in space. Types of insects consumed are discussed. Hazards of insect ingestion are considered. Insect reproduction, requirements, and raw materials conversion are discussed. Nutrition properties and composition of insects are considered. Preparation of insects as human food is discussed.

  10. Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

    DEFF Research Database (Denmark)

    Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih

    2002-01-01

    Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased....

  11. Evidence for ribosomal frameshifting and a novel overlapping gene in the genomes of insect-specific flaviviruses

    International Nuclear Information System (INIS)

    Firth, Andrew E.; Blitvich, Bradley J.; Wills, Norma M.; Miller, Cathy L.; Atkins, John F.

    2010-01-01

    Flaviviruses have a positive-sense, single-stranded RNA genome of ∼11 kb, encoding a large polyprotein that is cleaved to produce ∼10 mature proteins. Cell fusing agent virus, Kamiti River virus, Culex flavivirus and several recently discovered flaviviruses have no known vertebrate host and apparently infect only insects. We present compelling bioinformatic evidence for a 253-295 codon overlapping gene (designated fifo) conserved throughout these insect-specific flaviviruses and immunofluorescent detection of its product. Fifo overlaps the NS2A/NS2B coding sequence in the - 1/+ 2 reading frame and is most likely expressed as a trans-frame fusion protein via ribosomal frameshifting at a conserved GGAUUUY slippery heptanucleotide with 3'-adjacent RNA secondary structure (which stimulates efficient frameshifting in vitro). The discovery bears striking parallels to the recently discovered ribosomal frameshifting site in the NS2A coding sequence of the Japanese encephalitis serogroup of flaviviruses and suggests that programmed ribosomal frameshifting may be more widespread in flaviviruses than currently realized.

  12. Infections and endothelial cells

    NARCIS (Netherlands)

    Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

    2003-01-01

    Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

  13. Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

    Science.gov (United States)

    Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

    1991-08-01

    The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

  14. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

    Directory of Open Access Journals (Sweden)

    Joshua D Ramsay

    Full Text Available Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata, the intraleukocyte stage (schizont of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID, which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis

  15. Consuming insects

    NARCIS (Netherlands)

    Roos, N.; Huis, van A.

    2017-01-01

    How healthy are insects? This is a highly relevant question in view of the global interest in the potential of insects as a sustainable food source in food systems and diets. Edible insects, like other foods, can provide nutrients and dietary energy to meet the requirements of the human body as a

  16. Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Elena Ufimtseva

    2016-01-01

    Full Text Available The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells.

  17. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

    Science.gov (United States)

    Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

    2017-09-01

    Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

  18. Generation of a parvovirus B19 vaccine candidate.

    Science.gov (United States)

    Chandramouli, Sumana; Medina-Selby, Angelica; Coit, Doris; Schaefer, Mary; Spencer, Terika; Brito, Luis A; Zhang, Pu; Otten, Gillis; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Settembre, Ethan C

    2013-08-20

    Parvovirus B19 is the causative agent of fifth disease in children, aplastic crisis in those with blood dyscrasias, and hydrops fetalis. Previous parvovirus B19 virus-like-particle (VLP) vaccine candidates were produced by co-infection of insect cells with two baculoviruses, one expressing wild-type VP1 and the other expressing VP2. In humans, the VLPs were immunogenic but reactogenic. We have developed new VLP-based parvovirus B19 vaccine candidates, produced by co-expressing VP2 and either wild-type VP1 or phospholipase-negative VP1 in a regulated ratio from a single plasmid in Saccharomyces cerevisiae. These VLPs are expressed efficiently, are very homogeneous, and can be highly purified. Although VP2 alone can form VLPs, in mouse immunizations, VP1 and the adjuvant MF59 are required to elicit a neutralizing response. Wild-type VLPs and those with phospholipase-negative VP1 are equivalently potent. The purity, homogeneity, yeast origin, and lack of phospholipase activity of these VLPs address potential causes of previously observed reactogenicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Infection and Proliferation of Giant Viruses in Amoeba Cells.

    Science.gov (United States)

    Takemura, Masaharu

    2016-01-01

    Acanthamoeba polyphaga mimivirus, the first discovered giant virus with genome size and particle size much larger than previously discovered viruses, possesses several genes for translation and CRISPER Cas system-like defense mechanism against virophages, which co-infect amoeba cells with the giant virus and which inhibit giant virus proliferation. Mimiviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their stargate structure. After infection, giant virion factories (VFs) form in amoeba cytoplasm, followed by DNA replication and particle formation at peripheral regions of VF. Marseilleviruses, the smallest giant viruses, infect amoeba cells by phagocytosis or endocytosis, form larger VF than Mimivirus's VF in amoeba cytoplasm, and replicate their particles. Pandoraviruses found in 2013 have the largest genome size and particle size among all viruses ever found. Pandoraviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their mouth-like apical pores. The proliferation of Pandoraviruses occurs along with nucleus disruption. New virions form at the periphery of the region formerly occupied by the amoeba cell nucleus.

  20. Potential Cellular Signatures of Viral Infections in Human Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    J. Mikovits

    2001-01-01

    Full Text Available Expression profiling of cellular genes was performed using a 10,000 cDNA human gene array in order to identify expression changes following chronic infection of human hematopoietic cells with Kapsosi’s Sarcoma -associated Virus (KSHV also known as Human Herpesvirus 8 (HHV8 and Human T cell leukemia virus-1 (HTLV-1. We performed cell-free {\\it in vitro} infection of primary bone marrow derived CD34+ cells using semi-purified HHV8 and a mature IL-2 dependent T cell line, KIT 225, using highly concentrated viral stocks prepared from an infectious molecular clone of HTLV-1. Thirty days post infection, mRNA was isolated from infected cultures and uninfected controls and submitted for microarray analysis. More than 400 genes were differentially expressed more than two-fold following HHV8 infection of primary bone marrow derived CD34+ cells. Of these 400, interferon regulatory factor 4 (IRF4, cyclin B2, TBP-associated factor, eukaryotic elongation factor and pim 2 were up-regulated more than 3.5 fold. In contrast, less than 100 genes were differentially expressed more than two-fold following chronic infection of a mature T cell line with HTLV-1. Of these, only cdc7 was up-regulated more than 3.5 fold. These data may provide insight into cellular signatures of infection useful for diagnosis of infection as well as potential targets for therapeutic intervention.

  1. Alterations in the nuclear proteome of HIV-1 infected T-cells

    International Nuclear Information System (INIS)

    DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

    2014-01-01

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

  2. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  3. Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System

    Directory of Open Access Journals (Sweden)

    Bo Lin

    2014-01-01

    Full Text Available The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  4. 6K2-induced vesicles can move cell to cell during turnip mosaic virus infection

    Directory of Open Access Journals (Sweden)

    Romain eGrangeon

    2013-12-01

    Full Text Available To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs. However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked towards the plasma membrane and were associated with plasmodesmata (PD. We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP to visualize how 6K2 move intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.

  5. Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

    Science.gov (United States)

    O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

    2018-04-15

    JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

  6. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

    Science.gov (United States)

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

    2017-01-01

    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  7. Transovarial Transmission of Rickettsia spp. and Organ-Specific Infection of the Whitefly Bemisia tabaci

    Science.gov (United States)

    Brumin, Marina; Levy, Maggie

    2012-01-01

    The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome. PMID:22660706

  8. All insects are equal, but some insects are more equal than others

    OpenAIRE

    Fischer, Arnout R.H.; Steenbekkers, L.P.A.

    2018-01-01

    Purpose: Lack of acceptance of insects as food is considered a barrier against societal adoption of the potentially valuable contribution of insects to human foods. An underlying barrier may be that insects are lumped together as one group, while consumers typically try specific insects. The purpose of this paper is to investigate the ways in which Dutch consumers, with and without insect tasting experience, are more or less willing to eat different insects. Design/methodology/approach: In a ...

  9. Ureaplasma parvum infection alters filamin a dynamics in host cells

    Directory of Open Access Journals (Sweden)

    Brown Mary B

    2011-04-01

    Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

  10. Translation of both 5'TOP and non-TOP host mRNAs continues into the late phase of Baculovirus infection

    NARCIS (Netherlands)

    Oers, van M.M.; Doitsidou, M.; Thomas, A.A.M.; Maagd, de R.A.; Vlak, J.M.

    2003-01-01

    Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2 from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a 5' terminal oligopyrimidine (TOP) tract classified the lepidopteran rpL15

  11. Data on atherosclerosis specific antibody conjugation to nanoemulsions

    Directory of Open Access Journals (Sweden)

    Geoffrey Prévot

    2017-12-01

    Full Text Available This article present data related to the publication entitled “Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging” (Prévot et al., 2017 [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs using a heterobifunctional linker (DSPE-PEG-maleimide. Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

  12. IL-15 STIMULATED NATURAL KILLER CELLS CLEAR HIV-1 INFECTED CELLS FOLLOWING LATENCY REVERSAL EX VIVO.

    Science.gov (United States)

    Garrido, Carolina; Abad-Fernandez, Maria; Tuyishime, Marina; Pollara, Justin J; Ferrari, Guido; Soriano-Sarabia, Natalia; Margolis, David M

    2018-03-28

    Current efforts towards HIV eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8 + T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of IL-15 treatment on NK cell function and the potential of stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-dependent cytotoxicity (ADCC), cytotoxicity, IFN-γ production and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and more importantly, IL-15-treated NK cells were able to clear latently HIV infected cells after exposure to vorinostat, a clinically relevant latency reversing agent. We also demonstrate that NK cells from HIV infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation towards future immunotherapies to clear persistent HIV infection using NK cells. IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential, and more importantly, clearing HIV infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication. Copyright © 2018 American Society for Microbiology.

  13. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

    International Nuclear Information System (INIS)

    Motohashi, Tomoko; Shimojima, Tsukasa; Fukagawa, Tatsuo; Maenaka, Katsumi; Park, Enoch Y.

    2005-01-01

    Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses

  14. Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

    International Nuclear Information System (INIS)

    Hoever, Gerold; Vogel, Jens-Uwe; Lukashenko, Polina; Hofmann, Wolf-Karsten; Komor, Martina; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2005-01-01

    In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies

  15. Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

    Science.gov (United States)

    McDermott, M J; Weber, E; Hunter, S; Stedman, K E; Best, E; Frank, G R; Wang, R; Escudero, J; Kuner, J; McCall, C

    2000-05-01

    An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that

  16. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Directory of Open Access Journals (Sweden)

    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  17. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    Science.gov (United States)

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  18. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

    Science.gov (United States)

    Doitsh, Gilad; Galloway, Nicole L. K.; Geng, Xin; Yang, Zhiyuan; Monroe, Kathryn M.; Zepeda, Orlando; Hunt, Peter W.; Hatano, Hiroyu; Sowinski, Stefanie; Muñoz-Arias, Isa; Greene, Warner C.

    2014-01-01

    The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of `anti-AIDS' therapeutics targeting the host rather than the virus.

  19. Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

    Science.gov (United States)

    Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

    2013-01-11

    Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

  20. The Immune Responses of the Animal Hosts of West Nile Virus: A Comparison of Insects, Birds, and Mammals

    Directory of Open Access Journals (Sweden)

    Laura R. H. Ahlers

    2018-04-01

    Full Text Available Vector-borne diseases, including arboviruses, pose a serious threat to public health worldwide. Arboviruses of the flavivirus genus, such as Zika virus (ZIKV, dengue virus, yellow fever virus (YFV, and West Nile virus (WNV, are transmitted to humans from insect vectors and can cause serious disease. In 2017, over 2,000 reported cases of WNV virus infection occurred in the United States, with two-thirds of cases classified as neuroinvasive. WNV transmission cycles through two different animal populations: birds and mosquitoes. Mammals, particularly humans and horses, can become infected through mosquito bites and represent dead-end hosts of WNV infection. Because WNV can infect diverse species, research on this arbovirus has investigated the host response in mosquitoes, birds, humans, and horses. With the growing geographical range of the WNV mosquito vector and increased human exposure, improved surveillance and treatment of the infection will enhance public health in areas where WNV is endemic. In this review, we survey the bionomics of mosquito species involved in Nearctic WNV transmission. Subsequently, we describe the known immune response pathways that counter WNV infection in insects, birds, and mammals, as well as the mechanisms known to curb viral infection. Moreover, we discuss the bacterium Wolbachia and its involvement in reducing flavivirus titer in insects. Finally, we highlight the similarities of the known immune pathways and identify potential targets for future studies aimed at improving antiviral therapeutic and vaccination design.

  1. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    Science.gov (United States)

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

  2. Aboveground endophyte affects root volatile emission and host plant selection of a belowground insect.

    Science.gov (United States)

    Rostás, Michael; Cripps, Michael G; Silcock, Patrick

    2015-02-01

    Plants emit specific blends of volatile organic compounds (VOCs) that serve as multitrophic, multifunctional signals. Fungi colonizing aboveground (AG) or belowground (BG) plant structures can modify VOC patterns, thereby altering the information content for AG insects. Whether AG microbes affect the emission of root volatiles and thus influence soil insect behaviour is unknown. The endophytic fungus Neotyphodium uncinatum colonizes the aerial parts of the grass hybrid Festuca pratensis × Lolium perenne and is responsible for the presence of insect-toxic loline alkaloids in shoots and roots. We investigated whether endophyte symbiosis had an effect on the volatile emission of grass roots and if the root herbivore Costelytra zealandica was able to recognize endophyte-infected plants by olfaction. In BG olfactometer assays, larvae of C. zealandica were more strongly attracted to roots of uninfected than endophyte-harbouring grasses. Combined gas chromatography-mass spectrometry and proton transfer reaction-mass spectrometry revealed that endophyte-infected roots emitted less VOCs and more CO2. Our results demonstrate that symbiotic fungi in plants may influence soil insect distribution by changing their behaviour towards root volatiles. The well-known defensive mutualism between grasses and Neotyphodium endophytes could thus go beyond bioactive alkaloids and also confer protection by being chemically less apparent for soil herbivores.

  3. A rapid method for titration of ascovirus infectivity.

    Science.gov (United States)

    Han, Ningning; Chen, Zishu; Wan, Hu; Huang, Guohua; Li, Jianhong; Jin, Byung Rae

    2018-05-01

    Ascoviruses are a recently described family and the traditional plaque assay and end-point PCR assay have been used for their titration. However, these two methods are time-consuming and inaccurate to titrate ascoviruses. In the present study, a quick method for the determination of the titer of ascovirus stocks was developed based on ascovirus-induced apoptosis in infected insect cells. Briefly, cells infected with serial dilutions of virus (10 -2 -10 -10 ) for 24 h were stained with trypan blue. The stained cells were counted, and the percentage of nonviable cells was calculated. The stained cell rate was compared between virus-infected and control cells. The minimum-dilution group that had a significant difference compared with control and the maximum-dilution group that had no significant difference were selected and then compared each well of the two groups with the average stained cell rate of control. The well was marked as positive well if the stained cell rate was higher than the average stained cell rate of control wells; otherwise, the well was marked as negative wells. The percentage of positive wells were calculated according to the number of positive. Subsequently, the virus titer was calculated through the method of Reed and Muench. This novel method is rapid, simple, reproducible, accurate, and less material-consuming and eliminates the subjectivity of the other procedures for titrating ascoviruses. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus

    OpenAIRE

    Maxwell, Philip W.; Chen, Genhui; Webster, John M.; Dunphy, Gary B.

    1994-01-01

    Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing t...

  5. Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

    Science.gov (United States)

    Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

    2012-01-01

    Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

  6. Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

    Science.gov (United States)

    Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

    2015-11-01

    Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p apoptosis at 12 hpi (p apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells.

  7. Functional and structural analysis of GP64, the major envelope glycoprotein of the Budded Virus phenotype of Autographa californica and Orgyia pseudotsugata Multicapsid Nucleopolyhedroviruses

    NARCIS (Netherlands)

    Oomens, A.G.P.

    1999-01-01

    The Baculoviridae are a family of large, enveloped, double-stranded DNA viruses, that cause severe disease in the larvae of mostly lepidopteran insects. Baculoviruses have been studied with the aim of developing alternatives to chemical pest control, and later for their potential as systems

  8. Insects and Scorpions

    Science.gov (United States)

    ... insects or scorpions can be hazardous to outdoor workers. Stinging or biting insects include bees, wasps, hornets, and fire ants. The health effects of stinging or biting insects or scorpions range ...

  9. Cells in Dengue Virus Infection In Vivo

    Directory of Open Access Journals (Sweden)

    Sansanee Noisakran

    2010-01-01

    Full Text Available Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF/dengue shock syndrome (DSS. The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

  10. Apoptosis in HEp-2 cells infected with Ureaplasma diversum.

    Science.gov (United States)

    Amorim, Aline Teixeira; Marques, Lucas Miranda; Santos, Angelita Maria Oliveira Gusmão; Martins, Hellen Braga; Barbosa, Maysa Santos; Rezende, Izadora Souza; Andrade, Ewerton Ferraz; Campos, Guilherme Barreto; Lobão, Tássia Neves; Cortez, Beatriz Araujo; Monezi, Telma Alvez; Machado-Santelli, Glaucia Maria; Timenetsky, Jorge

    2014-09-04

    Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

  11. TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection.

    Science.gov (United States)

    Wang, Jing; Qiao, Luhua; Hou, Zhouhua; Luo, Guangxiang

    2017-01-15

    Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope. TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1

  12. Plant rhabdoviruses: new insights and research needs in the interplay of negative-strand RNA viruses with plant and insect hosts.

    Science.gov (United States)

    Mann, Krin S; Dietzgen, Ralf G

    2014-08-01

    Rhabdoviruses are taxonomically classified in the family Rhabdoviridae, order Mononegavirales. As a group, rhabdoviruses can infect plants, invertebrates and vertebrates. Plant cyto- and nucleorhabdoviruses infect a wide variety of species across both monocot and dicot families, including agriculturally important crops such as lettuce, wheat, barley, rice, maize, potato and tomato. Plant rhabdoviruses are transmitted by and replicate in hemipteran insects such as aphids (Aphididae), leafhoppers (Cicadellidae), or planthoppers (Delphacidae). These specific interactions between plants, viruses and insects offer new insights into host adaptation and molecular virus evolution. This review explores recent advances as well as knowledge gaps in understanding of replication, RNA silencing suppression and movement of plant rhabdoviruses with respect to both plant and insect hosts.

  13. In vivo infection of IgG-containing cells by Jembrana disease virus during acute infection

    International Nuclear Information System (INIS)

    Desport, Moira; Tenaya, I.W. Masa; McLachlan, Alexander; McNab, Tegan J.; Rachmat, Judhi; Hartaningsih, Nining; Wilcox, Graham E.

    2009-01-01

    Jembrana disease virus (JDV) is an unusual bovine lentivirus which causes a non-follicular proliferation of lymphocytes, a transient immunosuppression and a delayed humoral response in infected Bali cattle in Indonesia. A double-immunofluorescent labeling method was developed to identify the subset of mononuclear cells in which the viral capsid protein could be detected. Viral antigen was present in pleomorphic centroblast-like cells which were identified as IgG-containing cells, including plasma cells, in lymphoid tissues. There was no evidence of infection of CD3 + T-cells or MAC387 + monocytes in tissues but large vacuolated cells with a macrophage-like morphology in the lung were found to contain viral antigen although they could not be shown conclusively to be infected. The tropism of JDV for mature IgG-containing cells may be relevant to understanding the pathogenesis of Jembrana disease, the delayed antibody responses and the genetic composition of this atypical lentivirus.

  14. Effect of gamma irradiation on proliferation and survival of Sf9 cells: radioresistance in a Lepidopteran insect cell line

    International Nuclear Information System (INIS)

    Seth, R.K.; Lovell, K.V.; Reynolds, S.E.

    2003-01-01

    Sf9 cells of Spodoptera frugiperda, when exposed to gamma-irradiation from a 60 Co source, were found markedly less sensitive to ionising radiation than mammalian cells in terms of both growth kinetics and survival. Following irradiation at 1.2 Gy S -1 there was a dose-dependent delay in Sf9 cell proliferation and plateau cell density was reduced. These effects were dependent on dose rate too. In the range 0.3 - 1.2 Gy s -1 , growth was delayed longer and reached a lower plateau with increasing dose rate. Exposure to radiation caused a decrease in adherence of cells to the substrate, and an increase in number of enlarged ('giant') cells. Analysis of colony formation after irradiation at 1.2 Gy s -1 gave a survival curve of conventional shape but with a very large D o value of 24 Gy. Extrapolation number (N) was 2.9, a value within the normal range for mammalian cells. At 0.12 Gy s -1 N had a similar value of 3.2, but D o was higher (30 Gy) than at the higher dose rate. This study indicates that the relative insensitivity of lepidoptera insects may be attributed to some extent to the intrinsic properties of their constituent cells. (author)

  15. Dendritic Cells Activate and Mature after Infection with Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Mamo Gezahagne

    2011-07-01

    Full Text Available Abstract Background Dendritic cells (DCs can take up an array of different antigens, including microorganisms which they can process and present more effectively than any other antigen presenting cell. However, whether the interaction between the human DC and Mycobacterium tuberculosis represents a defense mechanism by the invaded host, or helping the invader to evade the defense mechanism of the host is still not clearly understood. Findings To analyze the interactions between M. tuberculosis and immune cells, human peripheral blood monocyte-derived immature DCs were infected with M. tuberculosis H37Rv wild type strain and flow cytometry was used to analyse cell surface expression markers. The ability of the M. tuberculosis infected DC to induce T cell proliferation using 5 and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE dilution technique was also investigated. DCs were found to internalize the mycobacteria and show dose dependent infection and necrosis with different multiplicity of infection. Flow cytometry analysis of cell surface expression markers CD40, CD54, CD80, CD83, CD86 and HLA DR in infected DC revealed significant (p M. tuberculosis in comparison to immature DC with no stimulation. Lipopolysaccharide (LPS from Salmonella abortus equi, a known DC maturation agent, was used as a positive control and showed a comparable up regulation of cell surface markers as observed with M. tuberculosis infected DC. It was revealed that the M. tuberculosis infected DC induced T cell proliferation. Conclusion These data clearly demonstrate that M. tuberculosis induces activation and maturation of human monocyte-derived immature DC as well as induces T cell proliferation in vitro.

  16. Effect of rice husk biochar application to soil insect diversity on potato cultivation

    Science.gov (United States)

    Meilin, A.; Rubiana, R.

    2018-02-01

    High intensity of disease infection and the intensive use of fertilizers and pesticidescause saturated fertilizer and pesticide to the land. Remediation using biochar rice husk is one of the technology to decrease fertilizer and pesticide residue. The diversity of soil insects can be used as bioindicators because of their existence dependsg on soil structure and condition. This study was aimed to study the diversity and structure communities of soil insect in potatoes on difference husk rice biochar application. The sampling of soil insects was done on potato farmer’s land with four treatments i.e control (farmers’ technique), trichokompos without biochar, trichokompos + biochar with dose 1 ton/ha, and trichokompos + biochar with dose 2 ton / ha. At each point a single pitfall trap was installed for two nights and then it was taken for identification. The results showed that biochar application had significant effect on the number of soil insect species (P = 0.037). The soil insect species composition pattern also showed significant differences between the four treatments (R: 0.2306, Pvalue = 0.001). This mean that the application of biochar affects the number of insects species and plays a role in the formation of soil insect diversity beta patterns.

  17. Unconventional Pro-inflammatory CD4+ T Cell Response in B Cell-Deficient Mice Infected with Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Melisa Gorosito Serrán

    2017-11-01

    Full Text Available Chagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America but has become a global public health concern by migration of infected people. It has been reported that parasite persistence as well as the intensity of the inflammatory immune response are determinants of the clinical manifestations of the disease. Even though inflammation is indispensable for host defense, when deregulated, it can contribute to tissue injury and organ dysfunction. Here, we report the importance of B cells in conditioning T cell response in T. cruzi infection. Mice deficient in mature B cells (muMT mice infected with T. cruzi exhibited an increase in plasma TNF concentration, TNF-producing CD4+ T cells, and mortality. The increase in TNF-producing CD4+ T cells was accompanied by a reduction in IFNγ+CD4+ T cells and a decrease of the frequency of regulatory Foxp3+, IL-10+, and IL17+CD4+ T cells populations. The CD4+ T cell population activated by T. cruzi infection, in absence of mature B cells, had a high frequency of Ly6C+ cells and showed a lower expression of inhibitory molecules such as CTLA-4, PD-1, and LAG3. CD4+ T cells from infected muMT mice presented a high frequency of CD62LhiCD44− cells, which is commonly associated with a naïve phenotype. Through transfer experiments we demonstrated that CD4+ T cells from infected muMT mice were able to condition the CD4+ T cells response from infected wild-type mice. Interestingly, using Blimp-flox/flox-CD23icre mice we observed that in absence of plasmablast/plasma cell T. cruzi-infected mice exhibited a higher number of TNF-producing CD4+ T cells. Our results showed that the absence of B cells during T. cruzi infection affected the T cell response at different levels and generated a favorable scenario for unconventional activation of CD4+ T cell leading to an uncontrolled effector response and inflammation. The product of B cell differentiation, the plasmablast/plasma cells, could be able

  18. Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

    Science.gov (United States)

    Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

    2011-01-01

    Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

  19. A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

    Science.gov (United States)

    Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

    2017-08-01

    In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

  20. Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

    NARCIS (Netherlands)

    Geldmacher, Christof; Ngwenyama, Njabulo; Schuetz, Alexandra; Petrovas, Constantinos; Reither, Klaus; Heeregrave, Edwin J.; Casazza, Joseph P.; Ambrozak, David R.; Louder, Mark; Ampofo, William; Pollakis, Georgios; Hill, Brenna; Sanga, Erica; Saathoff, Elmar; Maboko, Leonard; Roederer, Mario; Paxton, William A.; Hoelscher, Michael; Koup, Richard A.

    2010-01-01

    HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common