WorldWideScience

Sample records for pseudorabies virus expressing

  1. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    OpenAIRE

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-01-01

    Abstract Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and...

  2. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

    Science.gov (United States)

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-06-16

    Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  3. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    Science.gov (United States)

    2011-01-01

    Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

  4. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    Directory of Open Access Journals (Sweden)

    Chen Dishi

    2011-06-01

    Full Text Available Abstract Background Porcine parvovirus (PPV VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs with similar morphology to the native capsid. Here, a pseudorabies virus (PRV system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28 following virulent PPV challenge compared with the control (7 of 31. Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  5. Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І.

    Science.gov (United States)

    Qian, Ping; Zhi, Xianwei; Wang, Bo; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

    2015-12-10

    Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype І (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains. The recombinant virus PRV TK(-)/gE(-)/PrM-E(+) which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK(-)/gE(-)/LacZ(+) digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK(-)/gE(-)/PrM-E(+) was further evaluated in mouse model. A recombinant pseudorabies virus (PRV TK(-)/gE(-)/PrM-E(+)) was successfully constructed. Mice experiments showed that PRV TK(-)/gE(-)/PrM-E(+) could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1 × 10(7) PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK(-)/gE(-)/PrM-E(+) vaccinated mice. PRV TK(-)/gE(-)/PrM-E(+) is a potential vaccine candidate against PRV and JEV GI infection in the future.

  6. Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

    Directory of Open Access Journals (Sweden)

    Petrovszki Pál

    2009-10-01

    Full Text Available Abstract Background Pseudorabies virus (PRV, a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection. Results In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners. Conclusion Most (if not all PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for

  7. Insertions in the gG Gene of Pseudorabies Virus Reduce Expression of the Upstream Us3 Protein and Inhibit Cell-to-Cell Spread of Virus Infection

    OpenAIRE

    Demmin, Gretchen L.; Clase, Amanda C.; Randall, Jessica A.; Enquist, L.W.; Banfield, Bruce W.

    2001-01-01

    The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar i...

  8. Biological characterization of a recombinant pseudorabies virus

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, E.; Prieto, C.; Martinez-Lobo, F. J.; Castro, J. M.

    2008-07-01

    In a previous study we obtained and characterized in vitro a novel pseudorabies virus (PRV) variant named gIp2 with a TK, gI/gE, 11k and 28k negative phenotype and a duplication of PK gene. The main objective of the present study was to determine the safety and efficacy, as a vaccine candidate, of this recombinant PRV. For this purpose, we used 24 PRV seronegative three weeks old piglets that were divided into five groups of treatment. Piglets of groups A and B were immunized twice with 10{sup 6}.5 and 10{sup 5}.5 TCID{sub 5}0 of gIp2, respectively; pigs of group C were vaccinated twice with MLV vaccine Auskipra GN and pigs of groups D and E were not immunized and served as infected and uninfected controls, respectively. Four weeks after the second immunization pigs of groups A, B, C and D were challenged by intranasal inoculation of 10{sup 6} TCID{sub 5}0 of the wild type NIA-3 strain of PRV. No adverse reactions or clinical signs were observed in any group after immunization, indicating that the application of up to 10 times the conventional dose included in a commercial vaccine (i.e. 10{sup 5}.5 TCID{sub 5}0) of gIp2 was safe in piglets. Additionally, the inoculation of gIp2 induced an immune response able to provide clinical and virological protection against pseudorabies virus after challenge. In conclusion, the use of gIp2 in piglets as a vaccine virus is safe and induces an immunity comparable to that exerted by commercially available vaccines. (Author) 34 refs.

  9. 9 CFR 113.213 - Pseudorabies Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... established as pure, safe, and immunogenic shall be used for preparing seeds for vaccine production. All... immunogenicity of vaccine prepared from the Master Seed in accordance with the Outline of Production shall be... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pseudorabies Vaccine, Killed Virus...

  10. Comparison of different prime-boost regimes with DNA and recombinant Orf virus based vaccines expressing glycoprotein D of pseudorabies virus in pigs.

    Science.gov (United States)

    van Rooij, E M A; Rijsewijk, F A M; Moonen-Leusen, H W; Bianchi, A T J; Rziha, H-J

    2010-02-17

    Both DNA and Orf virus (ORFV; Parapox virus) based vaccines have shown promise as alternatives for conventional vaccines in pigs against pseudorabies virus (PRV) infection causing Aujeszky's disease. In the present study we evaluated the efficacy of different prime-boost regimes in pigs in terms of immunogenicity and protection against challenge infection with PRV. The different prime-boost regimes consisted of the homologous prime-boost regimes (DNA followed by DNA or ORFV followed by ORFV) and the heterologous prime-boost regimes (DNA followed by ORFV and ORFV followed by DNA), all based on glycoprotein D (gD) of PRV. Moreover, we compared the efficacy of the different prime-boost regimes with the efficacy of a conventional modified live vaccine (MLV). The different prime-boost regimes resulted in different levels of immunity and protection against challenge infection. Most effective was the regime of priming with DNA vaccine followed by boosting with the ORFV based vaccine. This regime resulted in strong antibody responses, comparable to the antibody responses obtained after prime-boost vaccination with a conventional MLV vaccine. Also with regard to protection, the prime DNA-boost ORFV regime performed better than the other prime-boost regimes. This study demonstrates the potential of a heterologous prime-boost vaccination strategy against PRV based on a single antigen, and that in the natural host, the pig. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  11. Characterization and mapping of a nonessential pseudorabies virus glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Wathen, M.W.; Wathen, L.M.K.

    1986-04-01

    Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoproteins. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by the marker rescue of a gIII/sup -/ mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.

  12. Motor-coordination-dependent learning, more than others, is impaired in transgenic mice expressing pseudorabies virus immediate-early protein IE180.

    Directory of Open Access Journals (Sweden)

    Juan C López-Ramos

    Full Text Available The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96 was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning, object recognition, spatial orientation (water maze, startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.

  13. Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

    Directory of Open Access Journals (Sweden)

    Furlong RA

    2009-12-01

    Full Text Available Abstract Background Pseudorabies virus (PRV is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult. Results In this report, we have analyzed native host (piglets gene expression changes in response to acute pseudorabies virus infection of the brain and lung using a printed human oligonucleotide gene set from Illumina. A total of 210 and 1130 out of 23,000 transcript probes displayed differential expression respectively in the brain and lung in piglets after PRV infection (p-value Conclusion This is the first comprehensive analysis of the global transcriptional response of the native host to acute alphaherpesvirus infection. The differentially regulated genes reported here are likely to be of interest for the further study and understanding of host viral gene interactions.

  14. Regulation of pseudorabies virus gG glycoprotein gene promoter independently of pseudorabies immediate early IE180 protein.

    Science.gov (United States)

    Muñoz, A L; Torres, M; Martín, B; Lerma, L; Tabarés, E

    2010-04-01

    The pseudorabies virus (PRV) glycoprotein known as gG is generally regarded as an early protein, and the immediate early IE180 protein regulates its expression during infection. This study, however, provides evidence that although induction by IE180 is observed, the expression of a marker protein (EGFP), or gG itself, under the control of the gG promoter, can also occur independently of the expression of IE180. This result was demonstrated both with transient transfection assays using plasmids and with viral infections. In transient transfections, the expression under control of the gG promoter depends on the cell type and surprisingly, can be 1.3-fold higher than the expression under the control of the IE180 promoter in Hela Tet-Off cells. Recombinant PRV S3 was constructed by replacing gE in the PRV genome with a chimeric transgene, expressing EGFP under the control of the gG promoter. In PK15 cells infected with NIA-3 wild-type virus or with S3 recombinant virus, expression of gG PRV mRNA (or EGFP mRNA) under the control of the gG promoter in the presence of cycloheximide was detected by RT-PCR. This again indicates that some basal expression was produced in infected cells independently of IE180. This expression was augmented by IE180 protein in both plasmid transfections and viral infections.

  15. Detection of pseudorabies virus by duplex droplet digital PCR assay.

    Science.gov (United States)

    Ren, Meishen; Lin, Hua; Chen, Shijie; Yang, Miao; An, Wei; Wang, Yin; Xue, Changhua; Sun, Yinjie; Yan, Yubao; Hu, Juan

    2018-01-01

    Aujeszky's disease, caused by pseudorabies virus (PRV), has damaged the economy of the Chinese swine industry. A large number of PRV gene-deleted vaccines have been constructed based on deletion of the glycoprotein E ( gE) gene combined with other virulence-related gene deletions, such as thymidine kinase ( TK), whereas PRV wild-type strains contain an intact gE gene. We developed a sensitive duplex droplet digital PCR (ddPCR) assay to rapidly detect PRV wild-type isolates and gE gene-deleted viral vaccines. We compared this assay with a TaqMan real-time PCR (qPCR) using the same primers and probes. Both assays exhibited good linearity and repeatability; however, ddPCR maintained linearity at extremely low concentrations, whereas qPCR did not. Based on positive results for both gE and gB, the detection limit of ddPCR was found to be 4.75 copies/µL in contrast of 76 copies/µL for qPCR, showing that ddPCR provided a 16-fold improvement in sensitivity. In addition, no nonspecific amplification was shown in specificity testing, and the PRV wild-type was distinguished from a gE-deleted strain. The ddPCR was more sensitive when analyzing clinical serum samples. Thus, ddPCR may become an appropriate detection platform for PRV.

  16. Pseudorabies virus infection alters neuronal activity and connectivity in vitro.

    Directory of Open Access Journals (Sweden)

    Kelly M McCarthy

    2009-10-01

    Full Text Available Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV, infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural

  17. Pseudorabies virus US3 protein kinase mediates actin stress fiber breakdown

    NARCIS (Netherlands)

    Minnebruggen, van G.; Favoreel, H.W.; Jacobs, L.; Nauwynck, H.J.

    2003-01-01

    Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus,

  18. The Us3-encoded protein kinase from pseudorabies virus affects egress of virions from the nucleus

    NARCIS (Netherlands)

    Wagenaar, F.; Pol, J.M.A.; Peeters, B.; Gielkens, A.L.J.; Wind, de N.; Kimman, T.G.

    1995-01-01

    We examined the influence of inactivation of various genes located in the unique short (U(s)) region of pseudorabies virus on virus replication and assembly in porcine nasal mucosa explant cultures. The following strains were used: the virulent wild-type strain NIA-3, and strains derived from NIA-3

  19. Successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines.

    Science.gov (United States)

    Brockmeier, S I; Lager, K M; Mengeling, W L

    1997-01-01

    Three gilts were vaccinated with a NYVAC vaccinia recombinant expressing glycoprotein gD of pseudorabies virus (PRV) (NYVAC/gD). After farrowing, the piglets were allowed to nurse normally to obtain colostral immunity and then were divided into four groups, receiving NYVAC/gD, a NYVAC recombinant expressing glycoprotein gB of PRV (NYVAC/gB), an inactivated PRV vaccine (iPRV), or no vaccine. The piglets were vaccinated twice, three weeks apart beginning at approximately two weeks of age and later challenged with virulent PRV oronasally. Piglets that received NYVAC/gB or iPRV were the best protected based on lack of mortality, lower temperature responses, decreased weight loss and decreased viral shedding after challenge. These results indicate effective strategies for stimulating active immune response while still under the protection of maternal immunity.

  20. Long-term Cre-mediated Retrograde Tagging of Neurons Using a Novel Recombinant Pseudorabies Virus

    Directory of Open Access Journals (Sweden)

    Hassana eOyibo

    2014-09-01

    Full Text Available Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP. These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo.

  1. Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26.

    Science.gov (United States)

    Hogue, Ian B; Jean, Jolie; Esteves, Andrew D; Tanneti, Nikhila S; Scherer, Julian; Enquist, Lynn W

    2018-01-01

    Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular

  2. An approach to a FMD vaccine based on genetic engineered attenuated pseudorabies virus: one experiment using VP1 gene alone generates an antibody responds on FMD and pseudorabies in swine.

    Science.gov (United States)

    Qian, Ping; Li, Xiang-Min; Jin, Mei-Lin; Peng, Gui-Qing; Chen, Huan-Chun

    2004-06-02

    Foot-and-mouth disease (FMD) and pseudorabies (PR) are two important infectious diseases in swine. An attenuated pseudorabies virus (PRV) has been successfully used as a gene delivery vector for the development of live-viral vaccines. In this study, a recombinant PRV-VP1 virus was constructed by fusioning the VP1 gene of FMD virus in frame to the N-terminal sequence of the gG gene of PRV. To test the protective immunity, 15 FMDV sero-negative white swine were divided into three groups and immunized with the recombinant PRV-VP1 virus, commercial FMD vaccine and vector virus (TK(-)/gG(-)/LacZ(+)), respectively, and challenged intramuscularly with 20 minimal infecting doses (MID) of virulent type O FMDV 4 weeks after booster immunization. Swine vaccinated with PRV-VP1 acquired antibodies against both FMDV and PRV, however, anti-FMDV antibodies were much lower than those vaccinated with the commercial FMD vaccine. Our results suggested that the recombinant PRV-VP1 virus, which only expressed FMDV VP1 gene controlled by PRV gG promoter, could not protect swine from the challenge of 20 MID type O FMDV, but could delay and reduce the clinical symptoms of FMD.

  3. Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.

    Directory of Open Access Journals (Sweden)

    Andrea E Granstedt

    Full Text Available The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV, which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG. We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.

  4. Pseudorabies virus infection (Aujeszky's disease) in an Iberian lynx (Lynx pardinus) in Spain: a case report.

    Science.gov (United States)

    Masot, A Javier; Gil, María; Risco, David; Jiménez, Olga M; Núñez, José I; Redondo, Eloy

    2017-01-05

    The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In species other than suids infection is normally fatal. In these mammals, including carnivores, PRV typically causes serious neurologic disease. The endangered Iberian lynx (Lynx pardinus) is a wild feline endemic to south-western Europe (Iberian Peninsula). The Iberian lynx was found to be the world's most endangered felid species in 2002. In wild felines, PRV infection has only been previously reported once in a Florida panther in 1994. No seropositive lynxes have ever been found, nor has PRV been detected in dead Iberian lynxes to date. We describe the first reported case of pseudorabies in an Iberian lynx (Lynx pardinus). Pseudorabies was diagnosed in a young wild Iberian lynx from Extremadura (SW Spain) by histopathological examination, immunohistochemistry, polymerase chain reaction (PCR) and sequence analysis. Gross lesions included alopecia of the ventral neck, bloody gastro-intestinal contents and congestion of the brain. Histopathological analysis showed a moderate nonsuppurative meningoencephalitis with diffuse areas of demyelination, necrotizing gastritis and enteritis of the small intestine. Pseudorabies virus (PRV) antigen was found in neuronal and non-neuronal cells of the brain, tonsils, and gastric glandular epithelial cells by immunohistochemical analysis. The presence of the virus in the brain was confirmed by nested PCR. The sequence analysis of the 146 bp fragment (from the viral glycoprotein B gene) showed that the amplified sequence matched (with 100% identity) the PRV genome. Furthermore, specific DNA from glycoprotein D and E encoding-genes was detected by conventional and real-time PCR, respectively, confirming the latter that this infection was produced by a wild-type PRV strain. This study supports the suspicion that PRV could infect the Iberian lynx. The detection of PRV in a dead Iberian lynx suggests that the virus may have a

  5. Assembly of pseudorabies virus genome-based transfer vehicle carrying major antigen sites of S gene of transmissible gastroenteritis virus: potential perspective for developing live vector vaccines.

    Science.gov (United States)

    Yin, Jiechao; Ren, Xiaofeng; Tian, Zhijun; Li, Yijing

    2007-03-01

    Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.

  6. Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

    Science.gov (United States)

    Csabai, Zsolt; Takács, Irma F; Snyder, Michael; Boldogkői, Zsolt; Tombácz, Dóra

    2017-09-01

    Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

  7. Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant

    NARCIS (Netherlands)

    Rooij, van E.M.A.; Glansbeek, H.L.; Hilgers, L.A.T.; Lintelo, te E.G.; Visser, de Y.E.; Boersma, W.J.A.; Haagmans, B.L.; Bianchi, A.T.J.

    2002-01-01

    To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and

  8. 9 CFR 113.318 - Pseudorabies Vaccine.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pseudorabies Vaccine. 113.318 Section... Virus Vaccines § 113.318 Pseudorabies Vaccine. Pseudorabies Vaccine shall be prepared from virus-bearing... be used for preparing seeds for vaccine production. All serials of vaccine shall be prepared from the...

  9. [Construction of an infectious clone of pseudorabies virus strain ZJ genome maintained as a bacterial artificial chromosome].

    Science.gov (United States)

    Yin, Wen-Ling; Yin, Long-Bo; Ye, Wei-Cheng; Sun, Xue-Qiang; Yao, Huo-Chun; Zhang, Miao-Tao; Wang, Yi-Cheng; Zhang, Cun

    2010-07-01

    pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.

  10. Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication.

    Science.gov (United States)

    Wang, Yi-Ping; Huang, Li-Ping; Du, Wen-Juan; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-08-01

    RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Characterization of a replication-incompetent pseudorabies virus mutant lacking the sole immediate early gene IE180.

    Science.gov (United States)

    Wu, Brendan W; Engel, Esteban A; Enquist, Lynn W

    2014-11-11

    The alphaherpesvirus pseudorabies virus (PRV) encodes a single immediate early gene called IE180. The IE180 protein is a potent transcriptional activator of viral genes involved in DNA replication and RNA transcription. A PRV mutant with both copies of IE180 deleted was constructed 20 years ago (S. Yamada and M. Shimizu, Virology 199:366-375, 1994, doi:10.1006/viro.1994.1134), but propagation of the mutant depended on complementing cell lines that expressed the toxic IE180 protein constitutively. Recently, Oyibo et al. constructed a novel set of PRV IE180 mutants and a stable cell line with inducible IE180 expression (H. Oyibo, P. Znamenskiy, H. V. Oviedo, L. W. Enquist, A. Zador, Front. Neuroanat. 8:86, 2014, doi:10.3389/fnana.2014.00086), which we characterized further here. These mutants failed to replicate new viral genomes, synthesize immediate early, early, or late viral proteins, and assemble infectious virions. The PRV IE180-null mutant did not form plaques in epithelial cell monolayers and could not spread from primary infected neurons to second-order neurons in culture. PRV IE180-null mutants lacked the property of superinfection exclusion. When PRV IE180-null mutants infected cells first, subsequent superinfecting viruses were not blocked in cell entry and formed replication compartments in epithelial cells, fibroblasts, and neurons. Cells infected with PRV IE180-null mutants survived as long as uninfected cells in culture while expressing a fluorescent reporter gene. Transcomplementation with IE180 in epithelial cells restored all mutant phenotypes to wild type. The conditional expression of PRV IE180 protein enables the propagation of replication-incompetent PRV IE180-null mutants and will facilitate construction of long-term single-cell-infecting PRV mutants for precise neural circuit tracing and high-capacity gene delivery vectors. Pseudorabies virus (PRV) is widely used for neural tracing in animal models. The virus replicates and spreads between

  12. EVIDENCE OF PSEUDORABIES VIRUS SHEDDING IN FERAL SWINE ( SUS SCROFA) POPULATIONS OF FLORIDA, USA.

    Science.gov (United States)

    Hernández, Felipe A; Sayler, Katherine A; Bounds, Courtney; Milleson, Michael P; Carr, Amanda N; Wisely, Samantha M

    2018-01-01

    :  Feral swine ( Sus scrofa) are a pathogen reservoir for pseudorabies virus (PrV). The virus can be fatal to wildlife and contributes to economic losses in the swine industry worldwide. National surveillance efforts in the US use serology to detect PrV-specific antibodies in feral swine populations, but PrV exposure is not a direct indicator of pathogen transmission among conspecifics or to non-suid wildlife species. We measured antibody production and the presence of PrV DNA in four tissue types from feral swine populations of Florida, US. We sampled blood, nasal, oral, and genital swabs from 551 individuals at 39 sites during 2014-16. Of the animals tested for antibody production, 224 of 436 (51%) feral swine were antibody positive while 38 of 549 feral swine (7%) tested for viral shedding were quantitative polymerase chain reaction (qPCR)-positive for PrV. The detection of PrV DNA across all the collected sample types (blood, nasal, oral, and genital [vaginal] swabs) suggested viral shedding via direct (oronasal or venereal), and potentially indirect (through carcass consumption), routes of transmission among infected and susceptible animals. Fourteen of 212 seronegative feral swine were qPCR-positive, indicating 7% false negatives in the serologic assay. Our findings suggest that serology may underestimate the actual infection risk posed by feral swine to other species and that feral swine populations in Florida are capable of shedding the virus through multiple routes.

  13. Improved immune response to an attenuated pseudorabies virus vaccine by ginseng stem-leaf saponins (GSLS) in combination with thimerosal (TS).

    Science.gov (United States)

    Ni, Jingxuan; Bi, Shicheng; Xu, Wei; Zhang, Cenrong; Lu, Yisong; Zhai, Lijuan; Hu, Songhua

    2016-08-01

    Vaccination using attenuated vaccines remains an important method to control animal infectious diseases. The present study evaluated ginseng stem-leaf saponins (GSLS) and thimerosal (TS) for their adjuvant effect on an attenuated pseudorabies virus (aPrV) vaccine in mice. Compared to the group immunized with aPrV alone, the co-inoculation of GSLS and/or TS induced a higher antibody response. Particularly, when administered together with GSLS-TS, the aPrV vaccine provoked a higher serum gB-specific antibody, IgG1 and IgG2a levels, lymphocyte proliferative responses, as well as production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) from lymphocytes, and more importantly provided an enhanced cytotoxicity of NK cells and protection against virulent field pseudorabies virus challenge. Additionally, the increased expression of miR-132, miR-146a, miR-147 and miR-155 was found in murine macrophages cultured with GSLS and/or TS. These data suggest that GSLS-TS as adjuvant improve the efficacy of aPrV vaccine in mouse model and have potential for the development of attenuated viral vaccines. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus.

    Science.gov (United States)

    Guo, Jin-Chao; Tang, Yan-Dong; Zhao, Kuan; Wang, Tong-Yun; Liu, Ji-Ting; Gao, Jia-Cong; Chang, Xiao-Bo; Cui, Hong-Yu; Tian, Zhi-Jun; Cai, Xue-Hui; An, Tong-Qing

    2016-01-01

    Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.

  15. Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

    Science.gov (United States)

    Hogue, Ian B.; Bosse, Jens B.; Hu, Jiun-Ruey; Thiberge, Stephan Y.; Enquist, Lynn W.

    2014-01-01

    Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress. PMID:25474634

  16. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.

    Directory of Open Access Journals (Sweden)

    Ian B Hogue

    2014-12-01

    Full Text Available Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.

  17. An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant.

    Science.gov (United States)

    Wang, Jichun; Guo, Rongli; Qiao, Yongfeng; Xu, Mengwei; Wang, Zhisheng; Liu, Yamei; Gu, Yiqi; Liu, Chang; Hou, Jibo

    2016-12-07

    Since the end of 2011 an outbreak of pseudorabies affected Chinese pig herds that had been vaccinated with the commercial vaccine made of Bartha K61 strain. It is now clear that the outbreak was caused by an emergent PRV variant. Even though vaccines made of PRV Bartha K61 strain can confer certain cross protection against PRV variants based on experimental data, less than optimal clinical protection and virus shedding reduction were observed, making the control or eradication of this disease difficult. An infectious clone of PRV AH02LA strain was constructed to generate a gE deletion mutant PRV(LA-AB) strain. PRV(LA-AB) strain can reach a titer of 108.43 TCID50 /mL (50% tissue culture infectious dose) on BHK-21 cells. To evaluate the efficiency of the inactivated vaccine made of PRV(LA-AB) strain, thirty 3-week-old PRV-negative piglets were divided randomly into six groups for vaccination and challenge test. All five piglets in the challenge control showed typical clinical symptoms of pseudorabies post challenge. Sneezing and nasal discharge were observed in four and three piglets in groups C(vaccinated with inactivated PRV Bartha K61 strain vaccine) and D(vaccinated with live PRV Bartha K61 strain vaccine) respectively. In contrast, piglets in both groups A(vaccinated with inactivated PRV LA-AB strain vaccine) and B(vaccinated with inactivated PRV LA-AB strain vaccine with adjuvant) presented mild or no clinical symptoms. Moreover, viral titers detected via nasal swabs were approximately 100 times lower in group B than in the challenge control, and the duration of virus shedding (3-4 days) was shorter than in either the challenge control (5-10 days) or groups C and D (5-6 days). The infectious clone constructed in this study harbors the whole genome of the PRV variant AH02LA strain. The gE deletion mutant PRV(LA-AB)strain generated from PRV AH02LA strain can reach a high titer on BHK-21 cells. An inactivated vaccine of PRV LA-AB provides clinical protection

  18. Structure-function dissection of the Pseudorabies virus glycoprotein B fusion loops.

    Science.gov (United States)

    Vallbracht, Melina; Brun, Delphine; Tassinari, Matteo; Vaney, Marie-Christine; Pehau-Arnaudet, Gérard; Guardado-Calvo, Pablo; Haouz, Ahmed; Klupp, Barbara G; Mettenleiter, Thomas C; Rey, Felix A; Backovic, Marija

    2017-10-18

    Conserved across the Herpesviridae family, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomain of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the Pseudorabies virus (PrV) gB ectodomain, revealing a typical class III post-fusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct sub-regions of the lipid bilayer and their role for membrane interactions. We tested 15 gB variants for their ability to bind to liposomes, and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275 and Tyr276, which were essential for liposome binding and for fusion in a cellular and viral context, form a continuous hydrophobic patch at the gB trimer surface. Together with reported results from other alpha-herpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192 and Tyr276 form a rim that inserts into the more superficial, interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gB from beta- and gamma-herpesviruses suggest that this membrane-interaction mode is valid for gB from all herpesviruses.IMPORTANCE Herpesviruses are common human and animal pathogens, which infect cells by entering via fusion of viral and cellular membranes and which cause life-long and incurable infections. Central to the membrane fusion event for entry is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself into the target

  19. A live gI/gE-deleted pseudorabies virus (PRV) protects weaned piglets against lethal variant PRV challenge.

    Science.gov (United States)

    Yin, Yue; Xu, Zhiwen; Liu, Xiaowan; Li, Ping; Yang, Fan; Zhao, Jun; Fan, Yi; Sun, Xiangang; Zhu, Ling

    2017-08-01

    Emerging pseudorabies virus (PRV) variant has led to frequent outbreaks of PRV infection among Bartha-K61-vaccinated swine population in Chinese swine farms and caused high mortality in pigs of all age since late 2011. Here, we generated a gE/gI-deleted PRV (rPRVXJ-delgI/gE-EGFP) based on PRV variant strain (PRV-XJ) through homologous DNA recombination. Compared to parental strain, rPRVXJ-delgI/gE-EGFP showed similar growth kinetics in vitro. Its safety and immunogenicity were evaluated in weaned piglets. Our results showed that piglets immunized with rPRVXJ-delgI/gE-EGFP did not exhibit any clinical symptoms, and a high level of gB-specific antibody was detected. After lethal challenge with variant PRV (PRV-FJ strain), all vaccinated piglets survived without showing any clinical symptoms except slight fever within 7 days post-challenge. In unvaccinated piglets, typical clinical symptoms of pseudorabies were observed, and the piglets were all died at 5 days post-challenge. These results indicated that a live rPRVXJ-delgI/gE-EGFP vaccine could be a maker vaccine candidate to control the currently epidemic pseudorabies in China.

  20. Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine.

    Science.gov (United States)

    Hoegen, Bärbel; Saalmüller, Armin; Röttgen, Marlene; Rziha, Hanns-Joachim; Geldermann, Hermann; Reiner, Gerald; Pfaff, Eberhard; Büttner, Mathias

    2004-12-28

    In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of naïve PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.

  1. Detection of quantitative trait loci for resistance/susceptibility to pseudorabies virus in swine.

    Science.gov (United States)

    Reiner, Gerald; Melchinger, Elke; Kramarova, Marcela; Pfaff, Eberhardt; Büttner, Matthias; Saalmüller, Armin; Geldermann, Hermann

    2002-01-01

    This study describes genetic differences in resistance/susceptibility to pseudorabies virus (PrV) between European Large White and Chinese Meishan pigs, with a mapping of quantitative trait loci (QTL) obtained from a genome-wide scan in F(2) animals. Eighty-nine F(2) pigs were challenged intranasally at 12 weeks with 10(5) p.f.u. of the wild-type PrV strain NIA-3. For QTL analysis, 85 microsatellite markers, evenly spaced on the 18 porcine autosomes and on the pseudoautosomal region of the X chromosome, were genotyped. All pigs developed clinical signs, i.e. fever, from 3 to 7 days p.i. The pure-bred Large White pigs, the F(1) and three-quarters of the F(2) animals, but none of the Meishan pigs, developed neurological symptoms and died or were euthanized. QTLs for appearance/non-appearance of neurological symptoms were found on chromosomes 9, 5, 6 and 13. They explained 10.6-17.9% of F(2) phenotypic variance. QTL effects for rectal temperature after PrV challenge were found on chromosomes 2, 4, 8, 10, 11 and 16. Effects on chromosomes 9, 10 and 11 were significant on a genome-wide level. The results present chromosomal regions that are associated with presence/absence of neurological symptoms as well as temperature course after intranasal challenge with NIA-3. The QTLs are in proximity to important candidate genes that are assumed to play crucial roles in host defence against PrV.

  2. Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1)

    Czech Academy of Sciences Publication Activity Database

    Zouharová, D.; Lipenská, I.; Fojtiková, M.; Kulich, P.; Neca, J.; Slaný, M.; Kovařčík, K.; Turanek-Knotigová, P.; Hubatka, F.; Celechovská, H.; Mašek, J.; Koudelka, Š.; Procházka, L.; Eyer, L.; Plocková, J.; Bartheldyová, E.; Miller, A. D.; Růžek, Daniel; Raška, M.; Janeba, Zlatko; Turánek, J.

    2016-01-01

    Roč. 184, FEB 29 (2016), s. 84-93 ISSN 0378-1135 Institutional support: RVO:60077344 ; RVO:61388963 Keywords : Pseudorabies * Acyclic nucleoside phosphonates * DNA viruses * Cidofovir * Antiviral drugs * DNA polymerase Subject RIV: EE - Microbiology, Virology; CC - Organic Chemistry (UOCHB-X) Impact factor: 2.628, year: 2016

  3. A mouse model to study immunity against pseudorabies virus infection: Significance of CD4+ and CD8+ cells in protective immunity

    NARCIS (Netherlands)

    Bianchi, A.T.J.; Moonen-Leusen, H.W.M.; Milligen, van F.J.; Savelkoul, H.F.J.; Zwart, R.J.; Kimman, T.G.

    1998-01-01

    In this study we firstly established a vaccination/challenge model to study pseudorabies virus infection in mice. The mouse model was used to investigate the significance of CD4 and CD8 cells and of IFN production in protective immunity. Functional depletion of CD4 and CD8 and IFN was obtained in

  4. Pseudorabies Virus US3 Protein Kinase Protects Infected Cells from NK Cell-Mediated Lysis via Increased Binding of the Inhibitory NK Cell Receptor CD300a.

    Science.gov (United States)

    Grauwet, K; Vitale, M; De Pelsmaeker, S; Jacob, T; Laval, K; Moretta, L; Parodi, M; Parolini, S; Cantoni, C; Favoreel, H W

    2015-11-18

    Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this

  5. New model cell systems (PK and XTC-2) for studying acute and persistent infections with herpes simplex and pseudorabies viruses.

    Science.gov (United States)

    Szántó, J; Lesso, J; Golais, F

    1980-06-01

    Herpes simplex virus type 1 (HSV-1) showed limited replication in PK (pig kidney) and XTC-2 (Xenopus laevis frog) cell lines. Virus replication depended on the multiplicity of infection (MOI). At a high MOI, HSV-1 caused a typical cytopathic effect (CPE) in XTC-2 cells but a little marked CPE in PK cells. Pseudorabies virus (PRV) replicated intensively in PK cells (permissive system) but not in XTC-2 cells (nonpermissive system). Both viruses were adsorbed on to PK and XTC-2 cells. In infected PK cells, fluorescent HSV-1 antigen was demonstrated only in the vicinity of the nuclear membrane and in the paranuclear area of the cytoplasm but not in the nuclei. In XTC-2 cells, HSV-1 antigen was demonstrated also in the nuclei. Persistent HSV-1 infection was induced in PK but not in XTC-2 cells; it was of limited duration. PK cells which had lost HSV-1 multiplied further and proved susceptible to infection with HSV-1 or PRV.

  6. DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge.

    Science.gov (United States)

    Fischer, Laurent; Barzu, Simona; Andreoni, Christine; Buisson, Nathalie; Brun, André; Audonnet, Jean Christophe

    2003-04-02

    DNA vaccination represents a unique opportunity to overcome the limitations of conventional vaccine strategy in early life in the face of maternal-derived immunity. We used the model of pseudorabies virus (PRV) infection in pigs to further explore the potential of DNA vaccination in piglets born to sows repeatedly vaccinated with a PRV inactivated vaccine. A single immunisation of 8-week-old piglets with a DNA vaccine expressing secreted forms of PRV gB, gC, and gD, triggered an active serological response, confirming that DNA vaccination can over-ride significant residual maternal-derived immunity. A clear anamnestic response was evidenced when a secondary DNA vaccination was performed at 11 weeks of age, suggesting that DNA vaccination, performed in the face of passive immunity, elicited a strong humoral memory. We subsequently explored the potential of DNA vaccination in neonate piglets (5-6 days of age) in the face of very high titres of maternal antibodies and demonstrated that very high titres of passive antibodies selectively inhibited serological responses but not the establishment of potent memory responses. Finally, we demonstrated that DNA vaccination provided protection against an infectious PRV challenge at the end of the fattening period (i.e. at approximately 5 months of age). Collectively, our results pave the way for a new flexible vaccination program, which could ensure uninterrupted protection of fattening pigs over their entire economical life under field conditions.

  7. Different methods of real-time PCR for detection of pseudorabies virus

    Directory of Open Access Journals (Sweden)

    Carolina Kymie Vasques Nonaka

    Full Text Available ABSTRACT: Pseudorabies (PR is a highly contagious viral disease of great animal health and economic importance in swine industry. The aim of this study was to evaluate different genomic regions, real-time PCR chemistries and equipment for the molecular diagnosis of PR. Eight primer pairs targeting four genes (gB, gC, gE, gD, three different qPCR chemistries (SybrGreen, hydrolysis probes and plexor and two equipment (ABI7500, Rotorgene 3000 were evaluated. Oligonucleotides targeting gB using hydrolysis probes showed the best performance after evaluating efficiency (99%, the detection limit (10-1.5 TCID50 mL-1 and diagnostic sensitivity and; therefore, those primers were selected for performance verification factors such as repeatability, reproducibility and robustness (1.39% variance between days, 24% variance between analysts and 4.07% variance in analysis error. The qPCR standardized and validated in this research proved to be reliable for the diagnosis of PR and may be used in diagnostic laboratories that follow ISO 17025 and ISO 16140.

  8. Detection of pseudorabies virus DNA in the inner ear of intranasally infected BALB/c mice with nucleic acid hybridization in situ

    Energy Technology Data Exchange (ETDEWEB)

    Falser, N.; Bandtlow, I.; Haus, M.; Wolf, H.

    1986-01-01

    Evidence for the pathogenicity of pseudorabies virus for the auditory and vestibular organs of experimentally infected mice is presented. The authors demonstrate viral genomes in cells of the peripheral sensory organs, the nerve structures, and the affected areas of the brain in single sections from an entire cranium of an adult mouse. The data were obtained by an in situ hybridization technique adapted for use with fixed, plastic-embedded materials using /sup 3/H and /sup 125/I-labeled EBV. In contrast to conventional methods which use frozen sections, they were able to analyze cartilaginous and bony materials with high resolution.

  9. Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs.

    Directory of Open Access Journals (Sweden)

    Yi-Quan Wu

    Full Text Available Pseudorabies virus (PRV belongs to Alphaherpesvirinae subfamily that causes huge economic loss in pig industry worldwide. It has been recently demonstrated that many herpesviruses encode microRNAs (miRNAs, which play crucial roles in viral life cycle. However, the knowledge about PRV-encoded miRNAs is still limited. Here, we report a comprehensive analysis of both viral and host miRNA expression profiles in PRV-infected porcine epithelial cell line (PK-15. Deep sequencing data showed that the ∼4.6 kb intron of the large latency transcript (LLT functions as a primary microRNA precursor (pri-miRNA that encodes a cluster of 11 distinct miRNAs in the PRV genome, and 209 known and 39 novel porcine miRNAs were detected. Viral miRNAs were further confirmed by stem-loop RT-PCR and northern blot analysis. Intriguingly, all of these viral miRNAs exhibited terminal heterogeneity both at the 5' and 3' ends. Seven miRNA genes produced mature miRNAs from both arms and two of the viral miRNA genes showed partially overlapped in their precursor regions. Unexpectedly, a terminal loop-derived small RNA with high abundance and one special miRNA offset RNA (moRNA were processed from a same viral miRNA precursor. The polymorphisms of viral miRNAs shed light on the complexity of host miRNA-processing machinery and viral miRNA-regulatory mechanism. The swine genes and PRV genes were collected for target prediction of the viral miRNAs, revealing a complex network formed by both host and viral genes. GO enrichment analysis of host target genes suggests that PRV miRNAs are involved in complex cellular pathways including cell death, immune system process, metabolic pathway, indicating that these miRNAs play significant roles in virus-cells interaction of PRV and its hosts. Collectively, these data suggest that PRV infected epithelial cell line generates a diverse set of host miRNAs and a special cluster of viral miRNAs, which might facilitate PRV replication in cells.

  10. The protective immune response against Pseudorabies virus induced by DNA vaccination is impaired if the plasmid harbors a functional Porcine circovirus type 2 rep and origin of replication.

    Science.gov (United States)

    Faurez, Florence; Grasland, Béatrice; Béven, Véronique; Cariolet, Roland; Keranflec'h, André; Henry, Aurélie; Jestin, André; Dory, Daniel

    2012-12-01

    A plasmid rendered replicative in mammalian cells by inserting the Porcine circovirus 2 (PCV2) origin of replication and replicase gene (Ori-rep) has been previously constructed. The aim of the present study was to evaluate if the replication capacity of this plasmid could be advantageously used to improve the protective immunity induced by DNA vaccination. In this case we used the porcine Pseudorabies virus (PrV) DNA vaccination model. The replicative capacity of the DNA vaccine did not improve the protective immunity against PrV in pigs, but on the contrary the presence of the PCV2 Ori-rep sequence was harmful in the induction of this immunity compared to an equivalent but non-replicative DNA vaccine. In addition, the distribution and the persistence of the replicative and non-replicative plasmids inside the body were the same. This is the first study showing an in vivo deleterious effect of the replicative active PCV2 Ori-rep on the natural and specific protection against PrV infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Identification and Analysis of Novel Viral and Host Dysregulated MicroRNAs in Variant Pseudorabies Virus-Infected PK15 Cells.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available Pseudorabies (PR is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15 cells, we sequenced a small RNA (sRNA library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT, these novel viral miRNAs are throughout the PRV genome like β-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function.

  12. Production of monoclonal antibody against EP0 protein of pseudorabies virus and determination of its recognized epitope.

    Science.gov (United States)

    Xiang, Keyu; Cheng, Yi; Zhou, Meizhen; Sun, Leilei; Ji, Yikuan; Wang, Yu; Zhang, Baoshi; Luo, Yongwen; Ju, Chunmei

    2014-12-01

    Early protein 0 (EP0) is especially important for modulating PRV gene expression and reactivation from the latent state, but the mechanisms have not been elucidated. In this study, six monoclonal antibodies (MAbs) against EP0 protein of PRV were generated and their characterizations were investigated. Western blot analysis showed all six MAbs could react with immunizing antigen, but only 2B12 and 2C6 could react with native EP0 protein from PRV-infected cells. ELISA additivity tests revealed that at least three epitopes in EP0 were defined by six MAbs. The epitope recognized by MAb 2B12 was further identified in 287-292 aa of EP0 protein using a series of expressed overlapping peptides. These MAbs may provide valuable tools for further research of the functions of EP0 in PRV infection.

  13. A differential ELISA based on recombinant immunodominant epitopes of the gE gene of SHV-1 in a baculovirus-insect cell system to discriminate between pigs infected naturally with pseudorabies and vaccinated pigs.

    Science.gov (United States)

    Serena, María Soledad; Metz, Germán Ernesto; Corva, Santiago Gerardo; Mórtola, Eduardo Carlos; Echeverría, María Gabriela

    2011-02-01

    In the present study, the fragment corresponding to the immunodominant epitopes of the gE gene (gEpi) from the CL15 Argentinean strain of pseudorabies virus was expressed successfully in a baculovirus-insect cell system that contained the M6 gene of Bluetongue virus, which encodes the NS1 nonstructural protein. This protein has the ability to polymerize into highly immunogenic tubules inside infected cells that can be purified at large quantities by ultracentrifugation. Previously, the NS1 protein has been expressed by fusing it to sequences derived from viruses, such as human immunodeficiency virus type 1, hepatitis B virus, bovine leukemia virus, foot-and-mouth disease virus and influenza A virus. In the present study, a recombinant protein was obtained containing the gEpi fused to NS1 (NS1-gEpi) and used it as ELISA antigen for detection of anti-gE antibodies in order to discriminate between infected and vaccinated animals. This is the first report where gEpi was expressed in this particular baculovirus-insect cell system. © 2010 Elsevier B.V. All rights reserved.

  14. Parainfluenza virus 5 expressing the G protein of rabies virus protects mice after rabies virus infection.

    Science.gov (United States)

    Huang, Ying; Chen, Zhenhai; Huang, Junhua; Fu, ZhenFang; He, Biao

    2015-03-01

    Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) strain expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. 78 FR 23740 - Notice of Availability of a Swine Brucellosis and Pseudorabies Proposed Action Plan

    Science.gov (United States)

    2013-04-22

    ... Animal and Plant Health Inspection Service Notice of Availability of a Swine Brucellosis and Pseudorabies... swine brucellosis and pseudorabies available for public review and comment. This action will allow... a potential new approach to managing swine brucellosis and pseudorabies available for public review...

  16. 78 FR 9028 - Notice of Availability of a Swine Brucellosis and Pseudorabies Proposed Action Plan

    Science.gov (United States)

    2013-02-07

    ... Animal and Plant Health Inspection Service Notice of Availability of a Swine Brucellosis and Pseudorabies... approach to managing swine brucellosis and pseudorabies available for public review and comment. Swine brucellosis and pseudorabies have been eliminated from commercial swine herds within the United States, but...

  17. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    Science.gov (United States)

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  18. Vaccinia virus as an expression vector.

    Science.gov (United States)

    Talavera, A; Rodriguez, J M

    1992-01-01

    Vaccinia virus (Vv) is a member of the genus Orthopoxvirus, one of seven genera included in the family Poxviridae. Most of these viruses infect vertebrates (Orthopoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Parapoxvirus), but one genus, Entomopoxvirus, infects insects. It is interesting to note that the Fibroma and Mixoma viruses of the leporipoxvirus genus cause tumors in their hosts (rabbits), these being the only tumorigenic viruses in the family (1,2).

  19. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    Science.gov (United States)

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  20. Replication-competent fluorescent-expressing influenza B virus.

    Science.gov (United States)

    Nogales, Aitor; Rodríguez-Sánchez, Irene; Monte, Kristen; Lenschow, Deborah J; Perez, Daniel R; Martínez-Sobrido, Luis

    2016-02-02

    Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  2. Recombinant measles virus vaccine expressing the Nipah virus glycoprotein protects against lethal Nipah virus challenge.

    Directory of Open Access Journals (Sweden)

    Misako Yoneda

    Full Text Available Nipah virus (NiV is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G. Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi. Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.

  3. Recombinant measles virus vaccine expressing the Nipah virus glycoprotein protects against lethal Nipah virus challenge.

    Science.gov (United States)

    Yoneda, Misako; Georges-Courbot, Marie-Claude; Ikeda, Fusako; Ishii, Miho; Nagata, Noriyo; Jacquot, Frederic; Raoul, Hervé; Sato, Hiroki; Kai, Chieko

    2013-01-01

    Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.

  4. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    Science.gov (United States)

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-08-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

  5. Protective efficacy of a recombinant Newcastle disease virus expressing glycoprotein of vesicular stomatitis virus in mice

    OpenAIRE

    Zhang, Minmin; Ge, Jinying; Li, Xiaofang; Chen, Weiye; Wang, Xijun; Wen, Zhiyuan; Bu, Zhigao

    2016-01-01

    Background Vesicular stomatitis virus (VSV) causes severe losses to the animal husbandry industry. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of VSV (rL-VSV-G) was constructed and its pathogenicity and immune protective efficacy in mouse were evaluated. Results In pathogenicity evaluation test, the analysis of the viral distribution in mouse organs and body weight change showed that rL-VSV-G was safe in mice. In immune protection assay, the reco...

  6. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2011-01-01

    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  7. Immunological surveillance in a pseudorabies quarantined herd using gilts and their progeny as sentinels.

    Science.gov (United States)

    Williams, P P; Sacks, J M; Yang, P C; Pirtle, E C; Erickson, G A; Beran, G W

    1989-01-01

    Specific pathogen free gilts and their progeny were evaluated to use as sentinels in a pseudorabies virus (PRV) infected herd by immunologically monitoring for PRV seroconversions. Time intervals targeted were pre- and post-PRV vaccinations, herd exposure, and farrowing to finishing. Post-PRV vaccinations, gilts showed low PRV lymphocyte stimulation and humoral responses. Following herd exposure, control gilts PRV seroconverted and PRV vaccinated gilts increased (2 to 4 times) in virus neutralization (VN) titers. Sixty-seven percent (4/6) of the progeny from a control gilt were PRV seropositive at finishing. Progeny from PRV vaccinated gilts were depleted of passive immunity by week 7, and were seronegative until week 9. At finishing 47% (14/30) of them were PRV seropositive indicating exposure to PRV. The VN test was not sensitive enough to detect weak positive serums, noted as positives by latex agglutination (LA) test, ELISA, and Western blots. The gilts and progeny detected PRV, respectively, in the herd housing quarters and in the farrow to finish facilities. A strategy for future sentinel experimental surveillances using primarily the LA test is proposed.

  8. Construction of PVX virus-expression vector to express enterotoxin ...

    African Journals Online (AJOL)

    Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

  9. Recombinant measles viruses expressing respiratory syncytial virus proteins induced virus-specific CTL responses in cotton rats.

    Science.gov (United States)

    Yamaji, Yoshiaki; Nakayama, Tetsuo

    2014-07-31

    Respiratory syncytial virus (RSV) is a common cause of serious lower respiratory tract illnesses in infants. Natural infections with RSV provide limited protection against reinfection because of inefficient immunological responses that do not induce long-term memory. RSV natural infection has been shown to induce unbalanced immune response. The effective clearance of RSV is known to require the induction of a balanced Th1/Th2 immune response, which involves the induction of cytotoxic T lymphocytes (CTL). In our previous study, recombinant AIK-C measles vaccine strains MVAIK/RSV/F and MVAIK/RSV/G were developed, which expressed the RSV fusion (F) protein or glycoprotein (G). These recombinant viruses elicited antibody responses against RSV in cotton rats, and no infectious virus was recovered, but small amounts of infiltration of inflammatory cells were observed in the lungs following RSV challenge. In the present study, recombinant AIK-C measles vaccine strains MVAIK/RSV/M2-1 and MVAIK/RSV/NP were developed, expressing RSV M2-1 or Nucleoprotein (NP), respectively. These viruses exhibited temperature-sensitivity (ts), which was derived from AIK-C, and expressed respective RSV antigens. The intramuscular inoculation of cotton rats with the recombinant measles virus led to the induction of CD8(+) IFN-γ(+) cells. No infectious virus was recovered from a lung homogenate following the challenge. A Histological examination of the lungs revealed a significant reduction in inflammatory reactions without alveolar damage. These results support the recombinant measles viruses being effective vaccine candidates against RSV that induce RSV-specific CTL responses with or without the development of an antibody response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    Science.gov (United States)

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  11. A DNA vaccine expressing PB1 protein of influenza A virus protects mice against virus infection.

    Science.gov (United States)

    Košík, Ivan; Krejnusová, Ingrid; Práznovská, Margaréta; Poláková, Katarína; Russ, Gustáv

    2012-05-01

    Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.

  12. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    Energy Technology Data Exchange (ETDEWEB)

    Papaneri, Amy B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Wirblich, Christoph [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Cann, Jennifer A.; Cooper, Kurt [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Jahrling, Peter B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Schnell, Matthias J., E-mail: matthias.schnell@jefferson.edu [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jefferson Vaccine Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Blaney, Joseph E., E-mail: jblaney@niaid.nih.gov [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States)

    2012-12-05

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

  13. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and

  14. Interferon induction by viruses. XV. Biological characteristics of interferon induction-suppressing particles of vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Marcus, P.I.; Sekellick, M.J.

    1987-06-01

    A single interferon (IFN) induction-suppressing particle (ISP) of vesicular stomatitis virus (VSV) blocked completely the yield of IFN in a cell otherwise programmed to produce IFN. With mouse L cells as hosts, one lethal hit of UV radiation (D37 = 52.5 ergs/mm2) to the VSV genome sufficed to inactivate ISP activity; however, with ''aged'' primary chick embryo cells as hosts, it took 198 lethal hits (D37 = 10,395 ergs/mm2). ISP expression in chick cells did not require virus replication or amplified RNA synthesis, but did involve functional virion-associated L protein. ISP in chick cells also were capable of inhibiting, in a multiplicity-dependent manner, the plaquing efficiency of two viruses that require cellular polymerase II (pol II) for replication, e.g., pseudorabies and influenza. The refractory state to IFN inducibility that resulted from infection of chick cells with ISP (VSV tsO5 (UV = 100 hits)) was still extant after 6 days. In contrast, the plaquing efficiency of pseudorabies virus returned to control levels by 5 h after ISP infection. Chick cells infected with UV ISP remained viable, served as hosts for the replication of other viruses, and could be subcultured. Models are presented to account for these contrasting effects. The involvement of viral plus-strand leader RNA as an inhibitor of cellular pol II-dependent RNA synthesis, and the multifunctional activities of the virion-associated L protein, are discussed as possible molecules involved in the action of ISP in chick cells.

  15. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

    Science.gov (United States)

    Baek, Eseul; Yoon, Ju-Yeon; Palukaitis, Peter

    2017-10-01

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors

    Directory of Open Access Journals (Sweden)

    Lindbo John A

    2007-08-01

    Full Text Available Abstract Background Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use. Results We have constructed a Cauliflower mosaic virus (CaMV 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI. The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date. Conclusion These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.

  17. Lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses.

    Science.gov (United States)

    VanLeuven, James T; Ridenhour, Benjamin J; Gonzalez, Andres J; Miller, Craig R; Miura, Tanya A

    2017-01-01

    The severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. Disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. Murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. We used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, RV1B), moderate (coronavirus, MHV-1), and severe (influenza A virus, PR8) disease in mice. RV1B infection caused numerous gene expression changes, but the differential effect peaked at 12 hours post-infection. PR8 altered an intermediate number of genes whose expression continued to change through 24 hours. MHV-1 had comparatively few effects on host gene expression. The viruses elicited highly overlapping responses in antiviral genes, though MHV-1 induced a lower type I interferon response than the other two viruses. Signature genes were identified for each virus and included host defense genes for PR8, tissue remodeling genes for RV1B, and transcription factors for MHV-1. Our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract.

  18. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  19. Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

    Science.gov (United States)

    Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

    2013-01-01

    Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

  20. Cutting Edge: Innate Immune Augmenting Vesicular Stomatitis Virus Expressing Zika Virus Proteins Confers Protective Immunity.

    Science.gov (United States)

    Betancourt, Dillon; de Queiroz, Nina M G P; Xia, Tianli; Ahn, Jeonghyun; Barber, Glen N

    2017-04-15

    Zika virus (ZIKV) has become a serious public health concern because of its link to brain damage in developing human fetuses. Recombinant vesicular stomatitis virus (rVSV) was shown to be a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. In this study, we generated rVSVs (wild-type and attenuated VSV with mutated matrix protein [VSVm] versions) that express either the full length ZIKV envelope protein (ZENV) alone or include the ZENV precursor to the membrane protein upstream of the envelope protein, and our rVSV-ZIKV constructs showed efficient immunogenicity in murine models. We also demonstrated maternal protective immunity in challenged newborn mice born to female mice vaccinated with VSVm-ZENV containing the transmembrane domain. Our data indicate that rVSVm may be a suitable strategy for the design of effective vaccines against ZIKV. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

    Science.gov (United States)

    Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

    2014-12-05

    The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. 9 CFR 85.6 - Interstate movement of pseudorabies vaccinate swine, except swine from qualified negative gene...

    Science.gov (United States)

    2010-01-01

    ... vaccinate swine, except swine from qualified negative gene-altered vaccinated herds, not known to be..., except swine from qualified negative gene-altered vaccinated herds, not known to be infected with or exposed to pseudorabies. Pseudorabies vaccinate swine, except swine from qualified negative gene-altered...

  3. Varicella-Zoster Virus Expresses Multiple Small Noncoding RNAs.

    Science.gov (United States)

    Markus, Amos; Golani, Linoy; Ojha, Nishant Kumar; Borodiansky-Shteinberg, Tatiana; Kinchington, Paul R; Goldstein, Ronald S

    2017-12-15

    Many herpesviruses express small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), that may play roles in regulating lytic and latent infections. None have yet been reported in varicella-zoster virus (VZV; also known as human herpesvirus 3 [HHV-3]). Here we analyzed next-generation sequencing (NGS) data for small RNAs in VZV-infected fibroblasts and human embryonic stem cell-derived (hESC) neurons. Two independent bioinformatics analyses identified more than 20 VZV-encoded 20- to 24-nucleotide RNAs, some of which are predicted to have stem-loop precursors potentially representing miRNAs. These sequences are perfectly conserved between viruses from three clades of VZV. One NGS-identified sequence common to both bioinformatics analyses mapped to the repeat regions of the VZV genome, upstream of the predicted promoter of the immediate early gene open reading frame 63 (ORF63). This miRNA candidate was detected in each of 3 independent biological repetitions of NGS of RNA from fibroblasts and neurons productively infected with VZV using TaqMan quantitative PCR (qPCR). Importantly, transfected synthetic RNA oligonucleotides antagonistic to the miRNA candidate significantly enhanced VZV plaque growth rates. The presence of 6 additional small noncoding RNAs was also verified by TaqMan qPCR in productively infected fibroblasts and ARPE19 cells. Our results show VZV, like other human herpesviruses, encodes several sncRNAs and miRNAs, and some may regulate infection of host cells. IMPORTANCE Varicella-zoster virus is an important human pathogen, with herpes zoster being a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNAs) are recognized as important actors in modulating gene expression, and this study demonstrates the first reported VZV-encoded sncRNAs. Many are clustered to a small genomic region, as seen in other human herpesviruses. At least one VZV sncRNA was expressed in productive infection of neurons and fibroblasts

  4. Expression Profiles of Bovine Adeno-Associated Virus and Avian Adeno-Associated Virus Display Significant Similarity to That of Adeno-Associated Virus Type 5

    OpenAIRE

    Qiu, Jianming; Cheng, Fang; Pintel, David J.

    2006-01-01

    We present the first detailed expression profiles of nonprimate-derived adeno-associated viruses, namely, bovine adeno-associated virus (B-AAV) and avian adeno-associated virus (A-AAV), which were obtained after the infection of cell lines derived from their natural hosts. In general, the profiles of B-AAV and A-AAV were quite similar to that of AAV5; however, both exhibited features found for AAV2 as well. Like adeno-associated virus type 5 (AAV5), B-AAV and A-AAV utilized an internal polyad...

  5. Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets.

    Science.gov (United States)

    Marsh, Glenn A; Virtue, Elena R; Smith, Ina; Todd, Shawn; Arkinstall, Rachel; Frazer, Leah; Monaghan, Paul; Smith, Greg A; Broder, Christopher C; Middleton, Deborah; Wang, Lin-Fa

    2013-03-25

    Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses. A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells. Three recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia. Here, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.

  6. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

    Directory of Open Access Journals (Sweden)

    Lin Na-Sheng

    2007-09-01

    Full Text Available Abstract Background Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV, that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. Methods We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s of the capsid protein VP1 of foot-and-mouth disease virus (FMDV. The recombinant BaMV plasmid (pBVP1 was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164 of FMDV VP1. Results The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. Conclusion Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

  7. [Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor].

    Science.gov (United States)

    Li, Huiming; Qiu, Wei; Wang, Feng; Wei, Fang; Chen, Xiafang; Wu, Xiaobing; Huang, Qian

    2008-02-01

    A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

  8. Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

    Science.gov (United States)

    Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

    2015-01-01

    Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to

  9. Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

    Science.gov (United States)

    Croft, Nathan P.; Smith, Stewart A.; Wong, Yik Chun; Tan, Chor Teck; Dudek, Nadine L.; Flesch, Inge E. A.; Lin, Leon C. W.; Tscharke, David C.; Purcell, Anthony W.

    2013-01-01

    Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity. PMID:23382674

  10. Single-dose vaccination of a recombinant parainfluenza virus 5 expressing NP from H5N1 virus provides broad immunity against influenza A viruses.

    Science.gov (United States)

    Li, Zhuo; Gabbard, Jon D; Mooney, Alaina; Gao, Xiudan; Chen, Zhenhai; Place, Ryan J; Tompkins, S Mark; He, Biao

    2013-05-01

    Influenza viruses often evade host immunity via antigenic drift and shift despite previous influenza virus infection and/or vaccination. Vaccines that match circulating virus strains are needed for optimal protection. Development of a universal influenza virus vaccine providing broadly cross-protective immunity will be of great importance. The nucleoprotein (NP) of influenza A virus is highly conserved among all strains of influenza A viruses and has been explored as an antigen for developing a universal influenza virus vaccine. In this work, we generated a recombinant parainfluenza virus 5 (PIV5) containing NP from H5N1 (A/Vietnam/1203/2004), a highly pathogenic avian influenza (HPAI) virus, between HN and L (PIV5-NP-HN/L) and tested its efficacy. PIV5-NP-HN/L induced humoral and T cell responses in mice. A single inoculation of PIV5-NP-HN/L provided complete protection against lethal heterosubtypic H1N1 challenge and 50% protection against lethal H5N1 HPAI virus challenge. To improve efficacy, NP was inserted into different locations within the PIV5 genome. Recombinant PIV5 containing NP between F and SH (PIV5-NP-F/SH) or between SH and HN (PIV5-NP-SH/HN) provided better protection against H5N1 HPAI virus challenge than did PIV5-NP-HN/L. These results suggest that PIV5 expressing NP from H5N1 has the potential to be utilized as a universal influenza virus vaccine.

  11. Programmed ribosomal frameshift alters expression of west nile virus genes and facilitates virus replication in birds and mosquitoes.

    Directory of Open Access Journals (Sweden)

    Ezequiel Balmori Melian

    2014-11-01

    Full Text Available West Nile virus (WNV is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

  12. Borna disease virus in mice: host-specific differences in disease expression.

    OpenAIRE

    Rubin, S A; Waltrip, R W; Bautista, J R; Carbone, K M

    1993-01-01

    We developed a mouse model of Borna disease to facilitate immunopathogenesis research by adaptation of Borna disease virus to mice through serial passage in mouse brain tissue. Borna disease virus replication, antibody production, inflammation, and Borna disease expression in several different strains of mice were examined.

  13. [Differences of the regulation on the expression of mucin 1 induced by two single-strand RNA viruses, respiratory syncytial virus and influenza virus].

    Science.gov (United States)

    Lu, Xin; Ni, Shu-Yuan; Li, Yu-Sheng

    2012-11-01

    To investigate whether influenza virus (IFZ) could up-regulate the expression of mucin 1 (MUC1) which exists in epithelial cells of upper respiratory track to restrict the inflammation, as respiratory syncytial virus (RSV) does. Quantitative RT-PCR and Western Blot were performed to detect the expression level of MUC1 induced by two single-strand RNA viruses in A549 cell lines. HEp-2 and MDCK cells were used respectively to culture RSV and IFZ. At 24h post A549 cells infection with the same titer of RSV or IFZ, the total RNA was harvest, qRT-PCR was then performed to observe the expression level of MUC1 mRNA. Meanwhile, at 24 h and 48 h post A549 cells infection with the same titer of RSV or IFZ, the total protein and supernatant were collected respectively after cell lysis, Western Blot was then used to detect the expression level of MUC1. Results showed that RSV could up-regulate the expression of MUC1 in airway epithelial cells with a significant dose-effect correlation, whereas IFZ could not. This study firstly investigated the differences of the regulation on the expression of MUC1 induced by two single-strand RNA viruses, and demonstrated initially that the mechanism of IFZ self-limiting differed from RSV, which attributed to up-regulation of the expression level of MUC1.

  14. Expression and silencing of cowpea mosaic virus transgenes

    NARCIS (Netherlands)

    Sijen, T.

    1997-01-01

    Plant viruses are interesting pathogens because they can not exist without their hosts and exploit the plant machinery for their multiplication. Fundamental knowledge on viral processes is of great importance to understand, prevent and control virus infections which can cause drastic losses

  15. The expression of essential components for human influenza virus internalisation in Vero and MDCK cells.

    Science.gov (United States)

    Ugiyadi, Maharani; Tan, Marselina I; Giri-Rachman, Ernawati A; Zuhairi, Fawzi R; Sumarsono, Sony H

    2014-05-01

    MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.

  16. Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

    Directory of Open Access Journals (Sweden)

    Teppei Sugawara

    2016-11-01

    Full Text Available Abstract Background The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus. Results Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV, whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene. Conclusion We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

  17. Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds.

    Science.gov (United States)

    Sayler, Katherine A; Bigelow, Troy; Koster, Leo G; Swenson, Sabrina; Bounds, Courtney; Hernández, Felipe; Wisely, Samantha M

    2017-07-01

    Despite successful eradication of pseudorabies virus (PRV) from the commercial pig industry in the United States in 2004, large populations of feral swine in certain regions act as wildlife reservoirs for the virus. Given the threat of reintroduction of the virus into domestic herds, a rapid, reliable, easily implemented assay is needed for detection of PRV. Although a real-time PCR (rtPCR) assay exists, improvements in rtPCR technology and a greater understanding of the diversity of PRV strains worldwide require an assay that would be easier to implement, more cost effective, and more specific. We developed a single-tube, rapid rtPCR that is capable of detecting 10 copies of PRV glycoprotein B ( gB) DNA per 20-µL total volume reaction. The assay did not produce a false-positive in samples known to be negative for the virus. The assay was negative for genetically similar herpesviruses and other porcine viruses. Our assay is a highly specific and sensitive assay that is also highly repeatable and reproducible. The assay should be a useful tool for early detection of PRV in pigs in the case of a suspected introduction or outbreak situation.

  18. Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

    Science.gov (United States)

    Marino, Nalini; Illingworth, Sam; Kodialbail, Prithvi; Patel, Ashvin; Calderon, Hugo; Lear, Rochelle; Fisher, Kerry D; Champion, Brian R; Brown, Alice C N

    2017-01-01

    Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

  19. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

    Directory of Open Access Journals (Sweden)

    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

  20. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

    Directory of Open Access Journals (Sweden)

    Mari Hirvinen

    2016-01-01

    Full Text Available In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

  1. Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells.

    Science.gov (United States)

    Lin, Hui-Wen; Liu, Cheng-Wei; Yang, Deng-Jye; Chen, Ching-Chung; Chen, Shih-Yin; Tseng, Jung-Kai; Chang, Tien-Jye; Chang, Yuan-Yen

    2017-10-01

    Recent investigations have demonstrated that carotenoid extract of Dunaliella salina alga (Alga) contains abundant β-carotene and has good anti-inflammatory activities. Murine macrophage (RAW264.7 cells) was used to establish as an in vitro model of pseudorabies virus-induced reactive oxygen species (ROS) response. In this study, antioxidant activities of Alga were measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity assays, reducing power, and virus-induced ROS formation in RAW264.7 cells. Anti-inflammatory activities of Alga were assessed by its ability to inhibit the production of interleukin-6 and nitric oxide (NO) using enzyme-linked immunosorbent assay, then the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated by measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (p50 and p65), JAK, STAT-1/3, and suppressor of cytokine signaling 3 (SOCS3) by Western blotting. In addition, Alga inhibited virus replication by plaque assay. Our results showed that the Alga had high antioxidant activity, significantly reduced the virus-induced accumulation of ROS, and inhibited the levels of nitric oxide and interleukin-6. Further studies revealed that Alga also downregulated the gene and protein expressions of iNOS, COX-2, nuclear factor-κB (p50 and p65), and the JAK/STAT pathway. The inhibitory effects of Alga were similar to pretreatment with specific inhibitors of JAK and STAT-3 in pseudorabies virus -infected RAW264.7 cells. Alga enhanced the expression of SOCS3 to suppress the activity of the JAK/STAT signaling pathway in pseudorabies virus-infected RAW264.7 cells. In addition, Alga has decreased viral replication (p < 0.005) at an early stage. Therefore, our results demonstrate that Alga inhibits ROS, interleukin6, and nitric oxide production via suppression of the JAK/STAT pathways and enhanced the

  2. Complementing defective viruses that express separate paramyxovirus glycoproteins provide a new vaccine vector approach.

    Science.gov (United States)

    Chattopadhyay, Anasuya; Rose, John K

    2011-03-01

    Replication-defective vaccine vectors based on vesicular stomatitis virus (VSV) lacking its envelope glycoprotein gene (G) are highly effective in animal models. However, such ΔG vectors are difficult to grow because they require complementation with the VSV G protein. In addition, the complementing G protein induces neutralizing antibodies in animals and thus limits multiple vector applications. In the process of generating an experimental Nipah virus (a paramyxovirus) vaccine, we generated two defective VSVΔG vectors, each expressing one of the two Nipah virus (NiV) glycoproteins (G and F) that are both required for virus entry to host cells. These replication-defective VSV vectors were effective at generating NiV neutralizing antibody in mice. Most interestingly, we found that these two defective viruses could be grown together and passaged in tissue culture cells in the absence of VSV G complementation. This mixture of complementing defective viruses was also highly effective at generating NiV neutralizing antibody in animals. This novel approach to growing and producing a vaccine from two defective viruses could be generally applicable to vaccine production for other paramyxoviruses or for other viruses where the expression of at least two different proteins is required for viral entry. Such an approach minimizes biosafety concerns that could apply to single, replication-competent VSV recombinants expressing all proteins required for infection.

  3. Virus and cell RNAs expressed during Epstein-Barr virus replication.

    Science.gov (United States)

    Yuan, Jing; Cahir-McFarland, Ellen; Zhao, Bo; Kieff, Elliott

    2006-03-01

    Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was approximately 5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol. 74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-kappaB responsive genes including IkappaBalpha and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication.

  4. Hepatitis C virus expressing reporter tagged NS5A protein

    DEFF Research Database (Denmark)

    2014-01-01

    Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable...... growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5'UTR, or heterotypic 5'UTR (either of genotype 1a (strain H...

  5. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    Science.gov (United States)

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  6. Recent patents involving virus nucleotide sequences; host defense, RNA silencing and expression vector strategies.

    Science.gov (United States)

    Ahmad, Tauqeer; AbouHaidar, Mounir; Hefferon, Kathleen L

    2011-12-01

    Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.

  7. Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

    Science.gov (United States)

    He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

    2017-04-01

    Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V+) and uninfected (V-) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V+ compared with V- isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V+ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V+ and V- isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

  8. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

    Directory of Open Access Journals (Sweden)

    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  9. The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus.

    OpenAIRE

    Wang, Y.; Xiang, Z.; Pasquini, S; Ertl, H C

    1997-01-01

    An E1-deleted, replication-defective adenovirus recombinant of the human strain 5 expressing the rabies virus glycoprotein, termed Adrab.gp, was tested in young mice. Mice immunized at birth with the Adrab.gp construct developed antibodies to rabies virus and cytokine-secreting lymphocytes and were protected against subsequent challenge. Maternal immunity to rabies virus strongly interferes with vaccination of the offspring with a traditional inactivated rabies virus vaccine. The immune respo...

  10. Expression of the Epstein-Barr virus gp350/220 gene in rodent and primate cells.

    OpenAIRE

    Whang, Y.; Silberklang, M; Morgan, A; Munshi, S; Lenny, A B; Ellis, R.W.; Kieff, E

    1987-01-01

    The gene encoding the Epstein-Barr virus envelope glycoproteins gp350 and gp220 was inserted downstream of the cytomegalovirus immediate-early, Moloney murine leukemia virus, mouse mammary tumor virus, or varicella-zoster virus gpI promoters in vectors containing selectable markers. Host cell and recombinant vector systems were defined which enabled the isolation of rodent or primate cell clones which expressed gp350/220 in substantial quantities. Continued expression of gp350/220 required ma...

  11. [Rapid selection of recombinant orf virus expression vectors using green fluorescent protein].

    Science.gov (United States)

    Zhang, Jiachun; Guo, Xianfeng; Zhang, Min; Wu, Feifan; Peng, Yongzheng

    2016-01-01

    To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene. The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.

  12. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  13. Differential Mucin Expression by Respiratory Syncytial Virus and Human Metapneumovirus Infection in Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Ma. Del Rocío Baños-Lara

    2015-01-01

    Full Text Available Mucins (MUC constitute an important component of the inflammatory and innate immune response. However, the expression of these molecules by respiratory viral infections is still largely unknown. Respiratory syncytial virus (RSV and human metapneumovirus (hMPV are two close-related paramyxoviruses that can cause severe low respiratory tract disease in infants and young children worldwide. Currently, there is not vaccine available for neither virus. In this work, we explored the differential expression of MUC by RSV and hMPV in human epithelial cells. Our data indicate that the MUC expression by RSV and hMPV differs significantly, as we observed a stronger induction of MUC8, MUC15, MUC20, MUC21, and MUC22 by RSV infection while the expression of MUC1, MUC2, and MUC5B was dominated by the infection with hMPV. These results may contribute to the different immune response induced by these two respiratory viruses.

  14. Expression of the herpes simplex virus type 1 latency-associated transcripts does not influence latency establishment of virus mutants deficient for neuronal replication.

    Science.gov (United States)

    Nicoll, M P; Efstathiou, S

    2013-11-01

    Herpes simplex virus type 1 establishes latency within neurons of the trigeminal ganglion. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs), which, whilst not essential for any aspect of latency, function to suppress lytic gene expression and enhance the survival of virus-infected neurons. The latent cell population comprises primary-order neurons infected directly from peripheral tissues and cells infected following further virus spread within the ganglion. In order to assess the role of LAT expression on latency establishment within first-order neurons, we infected ROSA26R reporter mice with Cre recombinase-expressing recombinant viruses harbouring deletion of the thymidine kinase lytic gene and/or the core LAT promoter. We found that LAT expression did not impact on latency establishment in viruses unable to replicate in neurons, and under these conditions, it was not required for the survival of neurons between 3 and 31 days post-infection.

  15. Avian adeno-associated virus-based expression of Newcastle disease virus hemagglutinin-neuraminidase protein for poultry vaccination.

    Science.gov (United States)

    Perozo, F; Villegas, P; Estevez, C; Alvarado, I R; Purvis, L B; Saume, E

    2008-06-01

    The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus member of the family Parvoviridae that has been proved to be useful as a viral vector for gene delivery. The use of AAAV for transgenic expression of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein and its ability to induce immunity in chickens were assessed. Proposed advantages of this system include no interference with maternal antibodies, diminished immune response against the vector, and the ability to accommodate large fragments of genetic information. In this work the generation of recombinant AAAV virions expressing the HN protein (rAAAV-HN) was demonstrated by electron microscopy, immunocytochemistry, and western blot analysis. Serological evidence of HN protein expression after in ovo or intramuscular inoculation of the recombinant virus in specific-pathogen-free chickens was obtained. Serum from rAAAV-HN-vaccinated birds showed a systemic immune response evidenced by NDV-specific enzyme-linked immunosorbent assay and hemagglutination inhibition testing. Positive virus neutralization in embryonated chicken eggs and indirect immunofluorescence detection of NDV infected cells by serum from rAAAV-HN vaccinated birds is also reported. A vaccine-challenge experiment in commercial broiler chickens using a Venezuelan virulent viscerotropic strain of NDV was performed. All unvaccinated controls died within 5 days postchallenge. Protection up to 80% was observed in birds vaccinated in ovo and revaccinated at 7 days of age with the rAAAV-HN. The results demonstrate the feasibility of developing and using an AAAV-based gene delivery system for poultry vaccination.

  16. Assessing the expression of chicken anemia virus proteins in plants

    NARCIS (Netherlands)

    Lacorte, C.C.; Lohuis, H.; Goldbach, R.W.; Prins, M.W.

    2007-01-01

    Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3 kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces

  17. Expression of VP60 gene from rabbit haemorrhagic disease virus ...

    African Journals Online (AJOL)

    The VP60 gene from rabbit haemorrhagic disease virus (RHDV) YL strain in Northeast of China, under control of the ats1A promoter from Rubisco small subunit genes of Arabidopsis thaliana, was introduced into the transfer deoxyribonucleic acid (T-DNA) region of plant transfer vector pCAMBIA1300 and transferred to ...

  18. Expression and Purification of Coat Protein of Citrus Tristeza Virus ...

    African Journals Online (AJOL)

    clearing, leaf mottling, leaf cupping, vein corking and symptomless are observed symptoms on infected plant ..... from a litter of culture is enough to immunize rabbit (one primary injection and six booster injection in ... The molecular basis for the antigenic diversity of CTV Implications for virus detection. Proc. Flo. State Hort.

  19. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector.

    Science.gov (United States)

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-03-18

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

  20. Faster replication and higher expression levels of viral glycoproteins give the vesicular stomatitis virus/measles virus hybrid VSV-FH a growth advantage over measles virus.

    Science.gov (United States)

    Ayala-Breton, Camilo; Russell, Luke O J; Russell, Stephen J; Peng, Kah-Whye

    2014-08-01

    VSV-FH is a hybrid vesicular stomatitis virus (VSV) with a deletion of its G glycoprotein and encoding the measles virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways. We studied the cytotoxic activity of a vesicular stomatitis/measles hybrid virus (VSV-FH), which is superior to that of measles virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and

  1. Attenuation of vaccinia virus by the expression of human Flt3 ligand

    Directory of Open Access Journals (Sweden)

    Sanda Miloslav

    2010-05-01

    Full Text Available Abstract Background Vaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt3 ligand (FL by recombinant vaccinia virus (rVACV highly influenced properties of the virus in dependence on the level of expression. Results High production of FL driven by the strong synthetic promoter decreased the growth of rVACV in macrophage cell line J774.G8 in vitro as well as its multiplication in vivo when inoculated in mice. The inhibition of replication in vivo was mirrored in low levels of antibodies against vaccinia virus (anti-VACV which nearly approached to the negative serum level in non-infected mice. Strong FL expression changed not only the host range of the recombinant but also the basic protein contents of virions. The major proteins - H3L and D8L - which are responsible for the virus binding to the cells, and 28 K protein that serves as a virulence factor, were changed in the membrane portion of P13-E/L-FL viral particles. The core virion fraction contained multiple larger, uncleaved proteins and a higher amount of cellular proteins compared to the control virus. The overexpression of FL also resulted in its incorporation into the viral core of P13-E/L-FL IMV particles. In contrary to the equimolar ratio of glycosylated and nonglycosylated FL forms found in cells transfected with the expression plasmid, the recombinant virus incorporated mainly the smaller, nonglycosylated FL. Conclusions It has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.

  2. Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

    Science.gov (United States)

    Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

    1985-01-01

    Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

  3. Gene Expression Profiling of Monkeypox Virus-Infected Cells Reveals Novel Interfaces for Host-Virus Interactions

    Science.gov (United States)

    2010-07-28

    2 days before infection with MPV. Culture medium was removed and cells were inoculated with crude monkey- pox virus-Katako Kombe strain (MPV-KK) at...morphology, cellular develop- ment, small molecule biochemistry, and posttransla- tional modification (Fig. 4B). The expression of histones exhibited...modulation have been described pre- viously, as in the indirect consequences of Ras, Rho, and Rab small GTPases regulation [87], its effect on viral

  4. Broad protection against avian influenza virus by using a modified vaccinia Ankara virus expressing a mosaic hemagglutinin gene.

    Science.gov (United States)

    Kamlangdee, Attapon; Kingstad-Bakke, Brock; Anderson, Tavis K; Goldberg, Tony L; Osorio, Jorge E

    2014-11-01

    A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4(+) and CD8(+) T cells were still detected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity. Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader immunity than a wild-type hemagglutinin. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Endogenous expression of proteases in colon cancer cells facilitate influenza A viruses mediated oncolysis.

    Science.gov (United States)

    Sturlan, Sanda; Stremitzer, Stefan; Bauman, Suzann; Sachet, Monika; Wolschek, Markus; Ruthsatz, Tanja; Egorov, Andrej; Bergmann, Michael

    2010-09-15

    Previously we have developed a prototype for conditionally replicating oncolytic influenza A virus which is based on deletions in the non-structural (NS1) protein. Multi-cycle replication of influenza A virus in malignant tissue is critically dependent on a protease which cleaves the viral entry protein. Here we demonstrate that the malignant colon cancer cell lines Caco-2, HT-29 and SW-620 can endogenously provide a virus-activating protease, which allows lytic multi-cycle replication of NS1 deletion viruses in those cancer cells in vitro. The oncolytic potency of an influenza NS1 deletion virus (NS1-80) was further tested in SCID mice bearing HT-29 derived tumors. The intra-tumoral injection of live, but not of heat inactivated NS1-80 virus significantly inhibited progression of established tumors. We conclude that a selected set of human cancer expressing virus activating- proteases will be a preferred target for oncolytic tumor therapy using influenza A virus mutants.

  6. Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Hongbing; Franz, Carl J.; Wu, Guang; Renshaw, Hilary; Zhao, Guoyan [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom); Wang, David, E-mail: davewang@borcim.wustl.edu [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States)

    2014-02-15

    Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.

  7. Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

    Science.gov (United States)

    Umadevi, P; Bhat, A I; Krishnamurthy, K S; Anandaraj, M

    2016-05-01

    Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies.

  8. Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins.

    Science.gov (United States)

    Hefferon, Kathleen Laura

    2012-11-10

    Transgenic plants present enormous potential as a cost-effective and safe platform for large-scale production of vaccines and other therapeutic proteins. A number of different technologies are under development for the production of pharmaceutical proteins from plant tissues. One method used to express high levels of protein in plants involves the employment of plant virus expression vectors. Plant virus vectors have been designed to carry vaccine epitopes as well as full therapeutic proteins such as monoclonal antibodies in plant tissue both safely and effectively. Biopharmaceuticals such as these offer enormous potential on many levels, from providing relief to those who have little access to modern medicine, to playing an active role in the battle against cancer. This review describes the current design and status of plant virus expression vectors used as production platforms for biopharmaceutical proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

    Science.gov (United States)

    Feenstra, Femke; van Gennip, René G P; Maris-Veldhuis, Mieke; Verheij, Eline; van Rijn, Piet A

    2014-09-01

    Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies. © 2014 The Authors.

  10. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Science.gov (United States)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  11. A Recombinant Antibody-Expressing Influenza Virus Delays Tumor Growth in a Mouse Model

    Directory of Open Access Journals (Sweden)

    Jennifer R. Hamilton

    2018-01-01

    Full Text Available Influenza A virus (IAV has shown promise as an oncolytic agent. To improve IAV as an oncolytic virus, we sought to design a transgenic virus expressing an immune checkpoint-inhibiting antibody during the viral life cycle. To test whether it was possible to express an antibody during infection, an influenza virus was constructed encoding the heavy chain of an antibody on the PB1 segment and the light chain of an antibody on the PA segment. This antibody-expressing IAV grows to high titers, and the antibodies secreted from infected cells exhibit comparable functionality with hybridoma-produced antibodies. To enhance the anti-cancer activity of IAV, an influenza virus was engineered to express a single-chain antibody antagonizing the immune checkpoint CTLA4 (IAV-CTLA4. In mice implanted with the aggressive B16-F10 melanoma, intratumoral injection with IAV-CTLA4 delayed the growth of treated tumors, mediated an abscopal effect, and increased overall survival.

  12. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

    Science.gov (United States)

    Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

    2017-11-24

    To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  13. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses

    Directory of Open Access Journals (Sweden)

    Yazmín Landeo-Ríos

    2017-11-01

    Full Text Available To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV (genus Crinivirus, family Closteroviridae encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV and Potato virus X (PVX, and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  14. Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

    Science.gov (United States)

    Feduchi, E; Aldabe, R; Novoa, I; Carrasco, L

    1995-12-15

    The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

  15. Optimization the expression of human papilloma virus E6 and E7 polytopic construct in E. coli expression system

    Directory of Open Access Journals (Sweden)

    Arian Rahimi

    2015-12-01

    Full Text Available Background: Human papilloma virus is a DNA virus from the papillomavirus family that is most prevalent in human cervical cancers and many studies showed the E6 and E7 proteins are present in the majority of cervical cancer cases. Development of universal HPV peptide-based vaccine with more serotypes coverage has considerable value. The aim of the study was to design a multi-epitope universal vaccine for major HPV based on E6 and E7 proteins and optimization the expression of polytopic construct contains E6 and E7 genes from different genotypes of human papilloma virus as a candid vaccine. Methods: In this experimental study that was carried out in Pasteur Institute of Iran, Virology Department from October 2013 to November 2014. In order to design the polytypic construct, we predicted the most probable immunogenic epitopes of E6 and E7 from common high risk HPV16, 18, 31, 45 along with high prevalent type 6 and 11 using bioinformatics methods. The synthetic pET28a expression vector harboring E6 and E7 protein was transformed into Escherichia coli hosts and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to expression optimization of recombinant protein, cell density, induction time, growth temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside concentration and cultures media were studied. Results: In the present study the recombinant fusion protein was expressed successfully and the highest expression of target protein was achieved in super broth medium containing 0.1% glucose and 0.2% L-arabinose. In Super broth medium, the optimum condition for recombinant protein expression was occurred at OD600 of 0.8, 0.1mM IPTG, one hour’s incubation time at 37 °C and BL21 (A1 host. Conclusion: The results of this study show that the optimum expression of E6 and E7 proteins from different genotypes of human papilloma virus can be performed. Moreover, by purification of recombinant protein and evaluation of its

  16. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

    Science.gov (United States)

    2006-04-21

    and adaptive immunity by Ebola and Lassa viruses . J. Immunol. 170:2797–2801. 30. Martini, G. A., and R. Siegert. 1971. Marburg virus disease...Immunol. 6:1191–1197. 41. Slenczka, W. G. 1999. The Marburg virus outbreak of 1967 and subsequent episodes. Curr. Top. Microbiol. Immunol. 235:49–75...Microbiology. All Rights Reserved. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

  17. Genome Wide Host Gene Expression Analysis in Chicken Lungs Infected with Avian Influenza Viruses.

    Directory of Open Access Journals (Sweden)

    Pradip B Ranaware

    Full Text Available The molecular pathogenesis of avian influenza infection varies greatly with individual bird species and virus strain. The molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV or the low pathogenic avian influenza virus (LPAIV infection in avian species remains poorly understood. Thus, global immune response of chickens infected with HPAI H5N1 (A/duck/India/02CA10/2011 and LPAI H9N2 (A/duck/India/249800/2010 viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAI H5N1 virus induced excessive expression of type I IFNs (IFNA and IFNG, cytokines (IL1B, IL18, IL22, IL13, and IL12B, chemokines (CCL4, CCL19, CCL10, and CX3CL1 and IFN stimulated genes (OASL, MX1, RSAD2, IFITM5, IFIT5, GBP 1, and EIF2AK in lung tissues. This dysregulation of host innate immune genes may be the critical determinant of the severity and the outcome of the influenza infection in chickens. In contrast, the expression levels of most of these genes was not induced in the lungs of LPAI H9N2 virus infected chickens. This study indicated the relationship between host immune genes and their roles in pathogenesis of HPAIV infection in chickens.

  18. Ceftiofur hydrochloride affects the humoral and cellular immune response in pigs after vaccination against swine influenza and pseudorabies.

    Science.gov (United States)

    Pomorska-Mól, Małgorzata; Czyżewska-Dors, Ewelina; Kwit, Krzysztof; Wierzchosławski, Karol; Pejsak, Zygmunt

    2015-10-22

    significant delay in the development of humoral response against pseudorabies virus (PRV) as well as a significant suppression of production of antibodies against swine influenza virus (SIV) was found in pigs receiving ceftiofur hydrochloride at the time of vaccination. The cellular immune response against PRV was also significantly affected by ceftiofur. In contrast, there were no significant differences between vaccinated groups with regard to the T-cell response against SIV. From day 28 of study to day 70, the concentration of INF-γ in culture supernatants were significantly lower in group treated with ceftiofur after restimulation with PRV. While, no significant differences were observed after restimulation of PBMC with H3N2 SIV. The effect of an antibiotic therapy with ceftiofur hydrochloride on the humoral and cellular post-vaccinal immune responses in pigs was investigated. Ceftiofur hydrochloride was given in therapeutic doses. The results of the present study indicate that both, humoral and cell-mediated post-vaccinal immune responses can be modulated by treatment with ceftiofur hydrochloride. The results of our study point out that caution should be taken when administered this antibiotic during vaccination of pigs.

  19. Genetic Inactivation of COPI Coatomer Separately Inhibits Vesicular Stomatitis Virus Entry and Gene Expression

    Science.gov (United States)

    Burdeinick-Kerr, Rebeca

    2012-01-01

    Viruses coopt cellular membrane transport to invade cells, establish intracellular sites of replication, and release progeny virions. Recent genome-wide RNA interference (RNAi) screens revealed that genetically divergent viruses require biosynthetic membrane transport by the COPI coatomer complex for efficient replication. Here we found that disrupting COPI function by RNAi inhibited an early stage of vesicular stomatitis virus (VSV) replication. To dissect which replication stage(s) was affected by coatomer inactivation, we used visual and biochemical assays to independently measure the efficiency of viral entry and gene expression in hamster (ldlF) cells depleted of the temperature-sensitive ε-COP subunit. We show that ε-COP depletion for 12 h caused a primary block to virus internalization and a secondary defect in viral gene expression. Using brefeldin A (BFA), a chemical inhibitor of COPI function, we demonstrate that short-term (1-h) BFA treatments inhibit VSV gene expression, while only long-term (12-h) treatments block virus entry. We conclude that prolonged coatomer inactivation perturbs cellular endocytic transport and thereby indirectly impairs VSV entry. Our results offer an explanation of why COPI coatomer is frequently identified in screens for cellular factors that support cell invasion by microbial pathogens. PMID:22072764

  20. N-Myc expression enhances the oncolytic effects of vesicular stomatitis virus in human neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Juan C Corredor

    2016-01-01

    Full Text Available N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB. Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVδM51 and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVδM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFN-stimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses.

  1. Expression of interferon gamma in the brain of cats with natural Borna disease virus infection.

    Science.gov (United States)

    Wensman, Jonas Johansson; Ilbäck, Carolina; Hjertström, Elina; Blomström, Anne-Lie; Gustavsson, Malin Hagberg; Jäderlund, Karin Hultin; Ström-Holst, Bodil; Belák, Sándor; Berg, Anna-Lena; Berg, Mikael

    2011-05-15

    Borna disease virus (BDV) is a neurotropic, negative-stranded RNA virus, which causes a non-suppurative meningoencephalomyelitis in a wide range of animals. In cats, BDV infection leads to staggering disease. In spite of a vigorous immune response the virus persists in the central nervous system (CNS) in both experimentally and naturally infected animals. Since the CNS is vulnerable to cytotoxic effects mediated via NK-cells and cytotoxic T-cells, other non-cytolytic mechanisms such as the interferon (IFN) system is favourable for viral clearance. In this study, IFN-γ expression in the brain of cats with clinical signs of staggering disease (N=12) was compared to the expression in cats with no signs of this disease (N=7) by quantitative RT-PCR. The IFN-γ expression was normalised against the expression of three reference genes (HPRT, RPS7, YWHAZ). Cats with staggering disease had significantly higher expression of IFN-γ compared to the control cats (p-value ≤ 0.001). There was no significant difference of the IFN-γ expression in BDV-positive (N=7) and -negative (N=5) cats having clinical signs of staggering disease. However, as BDV-RNA still could be detected, despite an intense IFN-γ expression, BDV needs to have mechanisms to evade this antiviral immune response of the host, to be able to persist. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging.

    Science.gov (United States)

    Padilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki; Melikyan, Gregory B

    2014-06-16

    The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

  3. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection

    DEFF Research Database (Denmark)

    Andersson, E C; Christensen, Jan Pravsgaard; Marker, O

    1994-01-01

    Virus-induced changes in adhesion molecule expression on T cells were investigated to understand how antiviral effector cells migrate into infectious foci. FACS analysis revealed that after systemic infection with lymphocytic choriomeningitis virus a number of cell adhesion molecules, including VLA......, it was found that up-regulation of VLA-4 expression on splenic T cells correlated with influx of inflammatory cells into the cerebrospinal fluid of intracerebrally infected animals, and that the number of CD8+VLA-4hi cells increased from lymph nodes and spleen to blood and cerebrospinal fluid. These results......-4, LFA-1, and ICAM-1, are up-regulated on CD8+ cells, whereas the lymph node homing receptor MEL-14 is down-regulated during the infection; only marginal changes were observed for CD4+ cells. Basically similar but less marked results were obtained in mice infected with Pichinde virus. Further...

  4. Production of Myxoma virus gateway entry and expression libraries and validation of viral protein expression.

    Science.gov (United States)

    Smallwood, Sherin E; Rahman, Masmudur M; Werden, Steven J; Martino, Maria Fernanda; McFadden, Grant

    2011-05-01

    Invitrogen's Gateway technology is a recombination-based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high-throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development.

  5. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  6. Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

    Science.gov (United States)

    James M. Slavicek; Nancy Hayes-Plazolles

    1991-01-01

    Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

  7. Potato virus Y induced changes in the gene expression of potato (Solanum tuberosum L.)

    NARCIS (Netherlands)

    Pompe-Novak, M.; Gruden, K.; Baebler, P.; Krecic-Stress, H.; Kovac, M.; Jongsma, M.A.; Ravnikar, M.

    2005-01-01

    The tuber necrotic strain of Potato virus Y (PVYNTN) causes potato tuber necrotic ringspot disease in sensitive potato cultivars. Gene expression in the disease response of the susceptible potato (Solanum tuberosum L.) cultivar Igor was investigated at different times after infection, using

  8. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  9. Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection.

    Science.gov (United States)

    Yeaman, Grant R; Howell, Alexandra L; Weldon, Sally; Demian, Douglas J; Collins, Jane E; O'Connell, Denise M; Asin, Susana N; Wira, Charles R; Fanger, Michael W

    2003-05-01

    Human immunodeficiency virus-1 (HIV-1) is primarily a sexually transmitted disease. Identification of cell populations within the female reproductive tract that are initially infected, and the events involved in transmission of infection to other cells, remain to be established. In this report, we evaluated expression of HIV receptors and coreceptors on epithelial cells in the uterus and found they express several receptors critical for HIV infection including CD4, CXCR4, CCR5 and galactosylceramide (GalC). Moreover, expression of these receptors varied during the menstrual cycle. Expression of CD4 and CCR5 on uterine epithelial cells is high throughout the proliferative phase of the menstrual cycle when blood levels of oestradiol are high. In contrast, CXCR4 expression increased gradually throughout the proliferative phase. During the secretory phase of the cycle when both oestradiol and progesterone are elevated, CD4 and CCR5 expression decreased whereas CXCR4 expression remained elevated. Expression of GalC on endometrial glands is higher during the secretory phase than during the proliferative phase of the menstrual cycle. Because epithelial cells line the female reproductive tract and express HIV receptors and coreceptors, it is likely that they are one of the first cell types to become infected. The hormonal regulation of HIV receptor expression may affect a woman's susceptibility to HIV infection during her menstrual cycle. Moreover, selective coreceptor expression could account for the preferential transmission of R5-HIV-1 strains to women. In addition, these studies provide evidence that the uterus, and potentially the entire upper reproductive tract, are important sites for the initial events involved in HIV infection.

  10. Expression and purification of coat protein of citrus tristeza virus ...

    African Journals Online (AJOL)

    Six colonies of TOP10 E. coli were selected and checked for the appropriate insertion of cp gene with PCR using T7F (5' TAA TAC GAC TCA CTA TAG GG 3') as forward primer and CTVCP2 as reverse primer. Two colonies having appropriate insertion were selected for transformation into BLD21 star (DE3) expression E.

  11. Ebola virus encodes a miR-155 analog to regulate importin-α5 expression.

    Science.gov (United States)

    Liu, Yuanwu; Sun, Jing; Zhang, Hongwen; Wang, Mingming; Gao, George Fu; Li, Xiangdong

    2016-10-01

    The 2014 outbreak of Ebola virus caused more than 10,000 human deaths. Current knowledge of suitable drugs, clinical diagnostic biomarkers and molecular mechanisms of Ebola virus infection is either absent or insufficient. By screening stem-loop structures from the viral genomes of four virulence species, we identified a novel, putative viral microRNA precursor that is specifically expressed by the Ebola virus. The sequence of the microRNA precursor was further confirmed by mining the existing RNA-Seq database. Two putative mature microRNAs were predicted and subsequently validated in human cell lines. Combined with this prediction of the microRNA target, we identified importin-α5, which is a key regulator of interferon signaling following Ebola virus infection, as one putative target. We speculate that this microRNA could facilitate the evasion of the host immune system by the virus. Moreover, this microRNA might be a potential clinical therapeutic target or a diagnostic biomarker for Ebola virus.

  12. Dynamic gene expression analysis in a H1N1 influenza virus mouse pneumonia model.

    Science.gov (United States)

    Bao, Yanyan; Gao, Yingjie; Shi, Yujing; Cui, Xiaolan

    2017-06-01

    H1N1, a major pathogenic subtype of influenza A virus, causes a respiratory infection in humans and livestock that can range from a mild infection to more severe pneumonia associated with acute respiratory distress syndrome. Understanding the dynamic changes in the genome and the related functional changes induced by H1N1 influenza virus infection is essential to elucidating the pathogenesis of this virus and thereby determining strategies to prevent future outbreaks. In this study, we filtered the significantly expressed genes in mouse pneumonia using mRNA microarray analysis. Using STC analysis, seven significant gene clusters were revealed, and using STC-GO analysis, we explored the significant functions of these seven gene clusters. The results revealed GOs related to H1N1 virus-induced inflammatory and immune functions, including innate immune response, inflammatory response, specific immune response, and cellular response to interferon-beta. Furthermore, the dynamic regulation relationships of the key genes in mouse pneumonia were revealed by dynamic gene network analysis, and the most important genes were filtered, including Dhx58, Cxcl10, Cxcl11, Zbp1, Ifit1, Ifih1, Trim25, Mx2, Oas2, Cd274, Irgm1, and Irf7. These results suggested that during mouse pneumonia, changes in the expression of gene clusters and the complex interactions among genes lead to significant changes in function. Dynamic gene expression analysis revealed key genes that performed important functions. These results are a prelude to advancements in mouse H1N1 influenza virus infection biology, as well as the use of mice as a model organism for human H1N1 influenza virus infection studies.

  13. A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein

    Science.gov (United States)

    Chen, Zhenhai; Zhou, Ming; Gao, Xiudan; Zhang, Guoqing; Ren, Guiping; Gnanadurai, Clement W.

    2013-01-01

    Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD50) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 106 PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 108 PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 108 PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines. PMID:23269806

  14. A novel rabies vaccine based on a recombinant parainfluenza virus 5 expressing rabies virus glycoprotein.

    Science.gov (United States)

    Chen, Zhenhai; Zhou, Ming; Gao, Xiudan; Zhang, Guoqing; Ren, Guiping; Gnanadurai, Clement W; Fu, Zhen F; He, Biao

    2013-03-01

    Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD(50)) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10(6) PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10(8) PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10(8) PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.

  15. Expression of measles virus nucleoprotein induces apoptosis and modulates diverse functional proteins in cultured mammalian cells.

    Directory of Open Access Journals (Sweden)

    Ashima Bhaskar

    Full Text Available BACKGROUND: Measles virus nucleoprotein (N encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. PRINCIPAL FINDINGS: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS was involved in the mechanism of cell death. Addition of ascorbic acid (AA or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. CONCLUSION: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.

  16. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L

    1999-01-01

    We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation...... of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount...

  17. Myxoma Virus Expressing Human Interleukin-12 Does Not Induce Myxomatosis in European Rabbits▿

    OpenAIRE

    Stanford, Marianne M.; Barrett, John W.; Gilbert, Philippe-Alexandre; Bankert, Richard; McFadden, Grant

    2007-01-01

    Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressi...

  18. [Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits].

    Science.gov (United States)

    Kuzmenko, Y V; Starodubova, E S; Karganova, G G; Timofeev, A V; Karpov, V L

    2016-01-01

    The interaction of viral proteins with host cell components plays an important role in antiviral immune response. One of the key steps of antiviral defense is the formation of immunoproteasomes. The effect of nonstructural protein 1 (NS1) of tick-borne encephalitis virus on the immunoproteasome formation was studied. It was shown that cell expression of NS1 does not reduce the efficacy of the immunoproteasome generation in response to interferon-γ stimulation and even increases the content of the immunoproteasome subunits without the interferon-γ treatment. Thus, NS1 of tick-borne encephalitis virus activates, rather than blocks the mechanisms of immune defense in the cell.

  19. Expression of infectious bovine rhinotracheitis virus glycoprotein D ...

    African Journals Online (AJOL)

    A 1047bp PCR product of the gD gene with EcoRI, HindIII restriction sites were subcloned of pTZ57R/T and digested by the mentioned endonucleases. Digested insert cloned in to pET-32a and transfered in E.coli cells. For the expression of gD protein, the pET-32a recombinant vector was transformed and then induced in ...

  20. Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

    Science.gov (United States)

    Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

    2013-01-01

    Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  1. Gene expression profiling of monkeypox virus-infected cells reveals novel interfaces for host-virus interactions

    Directory of Open Access Journals (Sweden)

    Ichou Mohamed

    2010-07-01

    Full Text Available Abstract Monkeypox virus (MPV is a zoonotic Orthopoxvirus and a potential biothreat agent that causes human disease with varying morbidity and mortality. Members of the Orthopoxvirus genus have been shown to suppress antiviral cell defenses, exploit host cell machinery, and delay infection-induced cell death. However, a comprehensive study of all host genes and virus-targeted host networks during infection is lacking. To better understand viral strategies adopted in manipulating routine host biology on global scale, we investigated the effect of MPV infection on Macaca mulatta kidney epithelial cells (MK2 using GeneChip rhesus macaque genome microarrays. Functional analysis of genes differentially expressed at 3 and 7 hours post infection showed distinctive regulation of canonical pathways and networks. While the majority of modulated histone-encoding genes exhibited sharp copy number increases, many of its transcription regulators were substantially suppressed; suggesting involvement of unknown viral factors in host histone expression. In agreement with known viral dependence on actin in motility, egress, and infection of adjacent cells, our results showed extensive regulation of genes usually involved in controlling actin expression dynamics. Similarly, a substantial ratio of genes contributing to cell cycle checkpoints exhibited concerted regulation that favors cell cycle progression in G1, S, G2 phases, but arrest cells in G2 phase and inhibits entry into mitosis. Moreover, the data showed that large number of infection-regulated genes is involved in molecular mechanisms characteristic of cancer canonical pathways. Interestingly, ten ion channels and transporters showed progressive suppression during the course of infection. Although the outcome of this unusual channel expression on cell osmotic homeostasis remains unknown, instability of cell osmotic balance and membrane potential has been implicated in intracellular pathogens egress. Our

  2. Expression of bioactive porcine interferon-alpha in Lactobacillus casei.

    Science.gov (United States)

    Ma, Shi-jie; Li, Kun; Li, Xin-Sheng; Guo, Xiao-Qing; Fu, Peng-Fei; Yang, Ming-Fan; Chen, Hong-Ying

    2014-09-01

    In this study, we constructed an expression cassette containing the inducible lac promoter and the secretion signal from an S-layer protein of Lactobacillus brevis for the expression of porcine interferon-alpha (IFN-α) in Lactobacillus casei (Lb. casei). Reverse-transcriptase PCR verified the presence of porcine IFN-α mRNA in the recombinant Lb. casei. The porcine IFN-α protein expressed in the recombinant Lb. casei was identified by both Western blot analysis and ELISA. We used various pH values and induction times to optimize the yield of IFN-α, and found that induction with 0.8% lactose for 16 h under anaerobic conditions produced the highest concentrations of IFN-α. Furthermore, the activity of porcine IFN-α in the cultural supernatant was evaluated on ST cells infected with pseudorabies virus. The results revealed that porcine IFN-α inhibited virus replication in vitro. The findings of our study indicate that recombinant Lb. casei producing porcine IFN-α has great potential for use as a novel oral antiviral agent in animal healthcare.

  3. Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

    Science.gov (United States)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

  4. Expression and partial characterisation of rabbit haemorrhagic disease virus non-structural proteins.

    Science.gov (United States)

    Urakova, Nadya; Frese, Michael; Hall, Robyn N; Liu, June; Matthaei, Markus; Strive, Tanja

    2015-10-01

    The intracellular replication and molecular virulence mechanisms of Rabbit haemorrhagic disease virus (RHDV) are poorly understood, mainly due to the lack of an effective cell culture system for this virus. To increase our understanding of RHDV molecular biology, the subcellular localisation of recombinant non-structural RHDV proteins was investigated in transiently transfected rabbit kidney (RK-13) cells. We provide evidence for oligomerisation of p23, and an ability of the viral protease to cleave the p16:p23 junction in trans, outside the context of the nascent polyprotein chain. Notably, expression of the viral polymerase alone and in the context of the entire RHDV polyprotein resulted in a redistribution of the Golgi network. This suggests that, similar to other positive-strand RNA viruses, RHDV may recruit membranes of the secretory pathway during replication, and that the viral polymerase may play a critical role during this process. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Myxoma Virus Expressing Interleukin-15 Fails To Cause Lethal Myxomatosis in European Rabbits▿

    Science.gov (United States)

    Liu, Jia; Wennier, Sonia; Reinhard, Mary; Roy, Edward; MacNeill, Amy; McFadden, Grant

    2009-01-01

    Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy. PMID:19279088

  6. Myxoma virus expressing interleukin-15 fails to cause lethal myxomatosis in European rabbits.

    Science.gov (United States)

    Liu, Jia; Wennier, Sonia; Reinhard, Mary; Roy, Edward; MacNeill, Amy; McFadden, Grant

    2009-06-01

    Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy.

  7. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

    Science.gov (United States)

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage

  8. Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors.

    Science.gov (United States)

    Kuo, Szu-Cheng; Chen, Ying-Ju; Wang, Yu-Ming; Kuo, Ming-Der; Jinn, Tzyy-Rong; Chen, Wen-Shuo; Chang, Yen-Chung; Tung, Kuo-Lun; Wu, Tzong-Yuan; Lo, Szecheng J

    2011-08-01

    Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

    Directory of Open Access Journals (Sweden)

    Maria R. Mendoza

    2017-11-01

    Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

  10. Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors.

    Science.gov (United States)

    Mendoza, Maria R; Payne, Alexandria N; Castillo, Sean; Crocker, Megan; Shaw, Brian D; Scholthof, Herman B

    2017-01-01

    Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies) it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV) referred to as TG, and Tobacco mosaic virus (TMV) annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

  11. Treatment of medulloblastoma with oncolytic measles viruses expressing the angiogenesis inhibitors endostatin and angiostatin.

    Science.gov (United States)

    Hutzen, Brian; Bid, Hemant Kumar; Houghton, Peter J; Pierson, Christopher R; Powell, Kimerly; Bratasz, Anna; Raffel, Corey; Studebaker, Adam W

    2014-03-19

    Medulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis. Oncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease. Medulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus' cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers of tumor-associated blood

  12. Intraventricular injection of myxoma virus results in transient expression of viral protein in mouse brain ependymal and subventricular cells.

    Science.gov (United States)

    France, Megan R; Thomas, Diana L; Liu, Jia; McFadden, Grant; MacNeill, Amy L; Roy, Edward J

    2011-01-01

    Oncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.

  13. Visualizing viral dissemination in the mouse nervous system, using a green fluorescent protein-expressing Borna disease virus vector.

    Science.gov (United States)

    Ackermann, Andreas; Guelzow, Timo; Staeheli, Peter; Schneider, Urs; Heimrich, Bernd

    2010-05-01

    Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.

  14. Primary EBV infection induces an expression profile distinct from other viruses but similar to hemophagocytic syndromes.

    Directory of Open Access Journals (Sweden)

    Samantha K Dunmire

    Full Text Available Epstein-Barr Virus (EBV causes infectious mononucleosis and establishes lifelong infection associated with cancer and autoimmune disease. To better understand immunity to EBV, we performed a prospective study of natural infection in healthy humans. Transcriptome analysis defined a striking and reproducible expression profile during acute infection but no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections, including influenza A (influenza, respiratory syncytial virus (RSV, human rhinovirus (HRV, attenuated yellow fever virus (YFV, and Dengue fever virus (DENV, revealed similarity only to DENV. The signature shared by EBV and DENV was also present in patients with hemophagocytic syndromes, suggesting these two viruses cause uncontrolled inflammatory responses. Interestingly, while EBV induced a strong type I interferon response, a subset of interferon induced genes, including MX1, HERC5, and OAS1, were not upregulated, suggesting a mechanism by which viral antagonism of immunity results in a profound inflammatory response. These data provide an important first description of the response to a natural herpesvirus infection in humans.

  15. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  16. Comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals virus-specific regulation of immune response genes.

    Science.gov (United States)

    Bourquain, Daniel; Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2013-02-20

    Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell's gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells. Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection. Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus infections in certain host species.

  17. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    Science.gov (United States)

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

  18. Quantitative impact of immunomodulation versus oncolysis with cytokine-expressing virus therapeutics.

    Science.gov (United States)

    Kim, Peter S; Crivelli, Joseph J; Choi, Il-Kyu; Yun, Chae-Ok; Wares, Joanna R

    2015-08-01

    The past century's description of oncolytic virotherapy as a cancer treatment involving specially-engineered viruses that exploit immune deficiencies to selectively lyse cancer cells is no longer adequate. Some of the most promising therapeutic candidates are now being engineered to produce immunostimulatory factors, such as cytokines and co-stimulatory molecules, which, in addition to viral oncolysis, initiate a cytotoxic immune attack against the tumor. This study addresses the combined effects of viral oncolysis and T-cell-mediated oncolysis. We employ a mathematical model of virotherapy that induces release of cytokine IL-12 and co-stimulatory molecule 4-1BB ligand. We found that the model closely matches previously published data, and while viral oncolysis is fundamental in reducing tumor burden, increased stimulation of cytotoxic T cells leads to a short-term reduction in tumor size, but a faster relapse. In addition, we found that combinations of specialist viruses that express either IL-12 or 4-1BBL might initially act more potently against tumors than a generalist virus that simultaneously expresses both, but the advantage is likely not large enough to replace treatment using the generalist virus. Finally, according to our model and its current assumptions, virotherapy appears to be optimizable through targeted design and treatment combinations to substantially improve therapeutic outcomes.

  19. DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells

    Directory of Open Access Journals (Sweden)

    Suresh V. Kuchipudi

    2015-06-01

    Full Text Available The data described in this article pertain to the article by Kuchipudi et al. (2014 titled “Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses” [1]. While infection of chickens with highly pathogenic avian influenza (HPAI H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEOdatabase with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014 [1].

  20. Expression, characterisation and antigenicity of a truncated Hendra virus attachment protein expressed in the protozoan host Leishmania tarentolae.

    Science.gov (United States)

    Fischer, Kerstin; dos Reis, Vinicius Pinho; Finke, Stefan; Sauerhering, Lucie; Stroh, Eileen; Karger, Axel; Maisner, Andrea; Groschup, Martin H; Diederich, Sandra; Balkema-Buschmann, Anne

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. [Experimental study on hepatitis B-virus X gene expression in adenoid cystic carcinoma].

    Science.gov (United States)

    Xie, Ling; Wang, Weihong; Xu, Biao; Liu, Yu

    2014-08-01

    To explore the expression of hepatitis B-virus X gene (HBX) in adenoid cystic carcinoma (ACC) and determine its clinical significance. Between June 2008 and October 2012, in-hospital patients with salivary gland tumors who were treated at the Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Kunming Medical University, were enrolled to this study. HBeAb-positive patients were defined as those exposed to hepatitis B virus (HBV) or harboring persistent HBV infection regardless of being HBeAg positive or negative. According to the pathological results, all patients were divided into ACC group and control group. Immunohistochemical staining and polymerase chain reaction (PCR) were used to detect HBX expression in ACC group and control group. HBX expression was mostly detected in the cytoplasm of ACC cells. Minimal HBX expression was detected in the nucleus. HBX expression was significantly higher in ACC than in Warthin's tumor. A significant difference was observed between the two groups. HBX is expressed in ACC and may be associated with the development of ACC. HBX might serve important functions in the carcinogenesis and development of ACC.

  2. Loss of P16 Protein Expression and Its Association with Epstein-Barr Virus LMP-1 Expression in Hodgkin's Lymphoma.

    Science.gov (United States)

    Irshaid, Fawzi; Tarawneh, Khaled; Alshdefat, Aisha; Dilmi, Fatiha; Jaran, Adnan; Al-Hadithi, Raji; Al-Khatib, Ahad

    2013-01-01

    Expression of Epstein-Barr virus Latent Member Protein-1 (EBV LMP-1) and loss of P16 protein expression are documented in lymphoma, indicating a relationship between them, but this relationship is not clear and sometimes contradictory. Thus, this study was conducted to examine the relationship between the loss of P16 and EBV LMP-1 expression in Jordanian patients diagnosed with lymphoma. Sections were made from archival formalin-fixed and paraffin-embedded blocks from 55 patients diagnosed with lymphoma. P16 expression and LMP-1 expression were detected by immunohistochemistry using monoclonal antibodies. In Hodgkin's Lymphoma (HL), the loss of P16 was higher in LMP-1 positive cases (61%) than LMP-1 negative cases (25%; P = 0.072). Conversely, in Non-Hodgkin's Lymphoma (NHL), none of LMP-1 positive samples showed loss of P16. Furthermore, among LMP-1 HL positive cases, the loss of P16 was more frequent in male (75%) than female (33%). Also, there was a significantly higher proportion of LMP-1 positive cases showing loss of P16 in HL (11:18), compared to those in NHL (0:8, P < 0.001), confirming a difference between HL and NHL, concerning the LMP-1/P16 relationship. A trend for an association between loss of P16 and LMP-1 expression was observed in HL but not NHL patients. These findings suggest that there are molecular and clinical differences in the pathogenesis and development of different subtypes of lymphoma.

  3. Virus-derived gene expression and RNA interference vector for grapevine.

    Science.gov (United States)

    Kurth, Elizabeth G; Peremyslov, Valera V; Prokhnevsky, Alexey I; Kasschau, Kristin D; Miller, Marilyn; Carrington, James C; Dolja, Valerian V

    2012-06-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

  4. Expression and Purification of Z Protein from Junín Virus

    Directory of Open Access Journals (Sweden)

    S. E. Goñi

    2010-01-01

    Full Text Available Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z. The arenavirus Junín (JUNV is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one. One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

  5. Myxoma Virus Expressing Human Interleukin-12 Does Not Induce Myxomatosis in European Rabbits▿

    Science.gov (United States)

    Stanford, Marianne M.; Barrett, John W.; Gilbert, Philippe-Alexandre; Bankert, Richard; McFadden, Grant

    2007-01-01

    Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressing the complete repertoire of MV proteins. Thus, vMyxIL-12 exhibits promise as an oncolytic candidate and is safe in all known vertebrate hosts, including lagomorphs. PMID:17728229

  6. Myxoma virus expressing human interleukin-12 does not induce myxomatosis in European rabbits.

    Science.gov (United States)

    Stanford, Marianne M; Barrett, John W; Gilbert, Philippe-Alexandre; Bankert, Richard; McFadden, Grant

    2007-11-01

    Myxoma virus (MV) is a candidate for oncolytic virotherapy due to its ability to selectively infect and kill tumor cells, yet MV is a species-specific pathogen that causes disease only in European rabbits. To assess the ability of MV to deliver cytokines to tumors, we created an MV (vMyxIL-12) that expresses human interleukin-12 (IL-12). vMyxIL-12 replicates similarly to wild-type MV, and virus-infected cells secrete bioactive IL-12. Yet, vMyxIL-12 does not cause myxomatosis, despite expressing the complete repertoire of MV proteins. Thus, vMyxIL-12 exhibits promise as an oncolytic candidate and is safe in all known vertebrate hosts, including lagomorphs.

  7. Engineering and expression of a RhoA peptide against respiratory syncytial virus infection in plants.

    Science.gov (United States)

    Ortega-Berlanga, Benita; Musiychuk, Konstantin; Shoji, Yoko; Chichester, Jessica A; Yusibov, Vidadi; Patiño-Rodríguez, Omar; Noyola, Daniel E; Alpuche-Solís, Ángel G

    2016-02-01

    MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide’s bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.

  8. Proteomic analysis of differentially expressed proteins in hepatitis B virus-related hepatocellular carcinoma tissues

    OpenAIRE

    Li, Ning; Long, Yunzhu; Fan, Xuegong; Liu, Hongbo; Li, Cui; Chen, Lizhang; Wang, Zhiming

    2009-01-01

    Abstract Background Hepatocellular carcinoma (HCC), a major cause of cancer death in China, is preceded by chronic hepatitis and liver cirrhosis (LC). Although hepatitis B virus (HBV) has been regarded as a clear etiology of human hepatocarcinogenesis, the mechanism is still needs to be further clarified. In this study, we used a proteomic approach to identify the differential expression protein profiles between HCC and the adjacent non-tumorous liver tissues. Methods Eighteen cases of HBV-re...

  9. Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Warke, Rajas V.; Xhaja, Kris; Martin, Katherine J.; Fournier, Marcia F.; Shaw, Sunil K.; Brizuela, Nathaly; de Bosch, Norma; Lapointe, David; Ennis, Francis A.; Rothman, Alan L.; Bosch, Irene

    2003-01-01

    Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance. PMID:14557666

  10. Recombinant rabies viruses expressing GM-CSF or flagellin are effective vaccines for both intramuscular and oral immunizations.

    Science.gov (United States)

    Zhou, Ming; Zhang, Guoqing; Ren, Guiping; Gnanadurai, Clement W; Li, Zhenguang; Chai, Qingqing; Yang, Yang; Leyson, Christina M; Wu, Wenxue; Cui, Min; Fu, Zhen F

    2013-01-01

    Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.

  11. Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

    Directory of Open Access Journals (Sweden)

    Dayton Andrew I

    2001-11-01

    Full Text Available Abstract Background Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. Results We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP, HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. Conclusions Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

  12. Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Chad E Mire

    Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

  13. Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection.

    Science.gov (United States)

    Babu, Mohan; Griffiths, Jonathan S; Huang, Tyng-Shyan; Wang, Aiming

    2008-07-09

    Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled. Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q or = 2.5 fold) and downregulated (viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection. Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions.

  14. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Davoud Koolivand

    2016-10-01

    Full Text Available The genomic region of Grapevine fanleaf virus (GFLV encoding the movement protein (MP was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3 to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG concentrations (1, 1.5, and 2 mM each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

  15. MicroRNA expression profiling of primary sheep testicular cells in response to bluetongue virus infection.

    Science.gov (United States)

    Du, Junzheng; Gao, Shandian; Tian, Zhancheng; Xing, Shanshan; Huang, Dexuan; Zhang, Guorui; Zheng, Yadong; Liu, Guangyuan; Luo, Jianxun; Chang, Huiyun; Yin, Hong

    2017-04-01

    Bluetongue virus (BTV) is a member of the genus Orbivirus within the family Reoviridae and causes a non-contagious, insect-transmitted disease in domestic and wild ruminants, mainly in sheep and occasionally in cattle and some species of deer. Virus infection can trigger the changes of the cellular microRNA (miRNA) expression profile, which play important post-transcriptional regulatory roles in gene expression and can greatly influence viral replication and pathogenesis. Here, we employed deep sequencing technology to determine which cellular miRNAs were differentially expressed in primary sheep testicular (ST) cells infected with BTV. A total of 25 known miRNAs and 240 novel miRNA candidates that were differentially expressed in BTV-infected and uninfected ST cells were identified, and 251 and 8428 predicted target genes were annotated, respectively. Nine differentially expressed miRNAs and their mRNA targets were validated by quantitative reverse transcription-polymerase chain reaction. Targets prediction and functional analysis of these regulated miRNAs revealed significant enrichment for several signaling pathways including MAPK, PI3K-Akt, endocytosis, Hippo, NF-kB, viral carcinogenesis, FoxO, and JAK-STAT signaling pathways. This study provides a valuable basis for further investigation on the roles of miRNAs in BTV replication and pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Vesicular stomatitis virus infection promotes immune evasion by preventing NKG2D-ligand surface expression.

    Directory of Open Access Journals (Sweden)

    Helle Jensen

    Full Text Available Vesicular stomatitis virus (VSV has recently gained attention for its oncolytic ability in cancer treatment. Initially, we hypothesized that VSV infection could increase immune recognition of cancer cells through induction of the immune stimulatory NKG2D-ligands. Here we show that VSV infection leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused an active suppression of NKG2D-ligand surface expression, affecting both endogenous and histone deacetylase (HDAC-inhibitor induced MICA, MICB and ULBP-2 expression. The classical immune escape mechanism of VSV (i.e., the M protein blockade of nucleocytoplasmic mRNA transport was not involved, as the VSV mutant strain, VSV(ΔM51, which possess a defective M protein, prevented MICA surface expression similarly to wild-type VSV. The VSV mediated down modulation of NKG2D-ligand expression did not involve apoptosis. Constitutive expression of MICA bypassed the escape mechanism, suggesting that VSV affect NKG2D-ligand expression at an early post-transcriptional level. Our results show that VSV possess an escape mechanism, which could affect the immune recognition of VSV infected cancer cells. This may also have implications for immune recognition of cancer cells after combined treatment with VSV and chemotherapeutic drugs.

  17. Evaluation of humoral and antigen-specific T-cell responses after vaccination of pigs against pseudorabies in the presence of maternal antibodies.

    Science.gov (United States)

    Pomorska-Mól, Małgorzata; Markowska-Daniel, Iwona; Pejsak, Zygmunt

    2010-08-26

    In this study the influence of maternal immunity against pseudorabies virus (PRV) on the development of humoral and T-cell mediated immune (CMI) responses was investigated under the experimental condition. Pigs born to immune sows were vaccinated with gE-deleted vaccine according to five different schedules. Peripheral blood mononuclear cells (PBMC), collected after vaccination, were used for PRV-induced lymphocyte proliferation assay (LPA). Antibodies to the gB and gE of PRV in serum were determined using ELISA kits. Maternally derived antibodies (MDA) in the serum of unvaccinated piglets born to immune sows were above the level considered to be positive until about 10-11 weeks of life. The active humoral as well as CMI responses was the highest in group vaccinated at 10 and 14 weeks of age. The results of this study suggest that MDA may disturb or even block development of active humoral response. Early priming of T-cells with attenuated gE-deleted PRV vaccine in the presence of MDA could be successful, but obtaining a long-term cellular immunity at least one booster is required. Copyright 2010 Elsevier B.V. All rights reserved.

  18. Use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.

    Directory of Open Access Journals (Sweden)

    Susan A Holechek

    Full Text Available Post-exposure vaccination with vaccinia virus (VACV has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV. Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

  19. Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon.

    Science.gov (United States)

    Barkhouse, Darryll A; Garcia, Samantha A; Bongiorno, Emily K; Lebrun, Aurore; Faber, Milosz; Hooper, D Craig

    2015-01-01

    Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR(-/-)) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons. Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the

  20. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.

    Science.gov (United States)

    Ball, L A

    1992-04-01

    RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

  1. Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies.

    Science.gov (United States)

    Poddar, Ankur; Ramasamy, Viswanathan; Shukla, Rahul; Rajpoot, Ravi Kant; Arora, Upasana; Jain, Swatantra K; Swaminathan, Sathyamangalam; Khanna, Navin

    2016-06-14

    Four antigenically distinct serotypes (1-4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype. We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies. This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs

  2. Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

    Science.gov (United States)

    MacKenzie, D J; Ellis, P J

    1992-01-01

    A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.

  3. The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

    Directory of Open Access Journals (Sweden)

    Galler R.

    1997-01-01

    Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

  4. Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

    Science.gov (United States)

    Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

    2017-10-15

    Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers.IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA

  5. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate.

    Science.gov (United States)

    Fontana, Diego; Kratje, Ricardo; Etcheverrigaray, Marina; Prieto, Claudio

    2015-08-20

    Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Inhibition of influenza A virus matrix and nonstructural gene expression using RNA interference.

    Science.gov (United States)

    McMillen, Cynthia M; Beezhold, Donald H; Blachere, Francoise M; Othumpangat, Sreekumar; Kashon, Michael L; Noti, John D

    2016-10-01

    Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influenza virus therapy. Published by Elsevier Inc.

  7. Differentially expressed genes in healthy and plum pox virus-infected Nicotiana benthamiana plants.

    Science.gov (United States)

    Vozárová, Z; Žilová, M; Šubr, Z

    2015-12-01

    Viruses use both material and energy sources of their hosts and redirect the production of disposable compounds in order to make viral replication more efficient. Metabolism of infected organisms is modified by these enhanced requirements as well by their own defense response. Resulting complex story consists of many regulation events on various gene expression levels. Elucidating these processes may contribute to the knowledge on virus-host interactions and to evolving new antiviral strategies. In our work we applied a subtractive cloning technique to compare the transcriptomes of healthy and plum pox virus (PPV)-infected Nicotiana benthamiana plants. Several genes were found to be induced or repressed by the PPV infection. The induced genes were mainly related to general stress response or photosynthesis, several repressed genes could be connected with growth defects evoked by the infection. Interestingly, some genes usually up-regulated by fungal or bacterial infection were found repressed in PPV-infected plants. Potential involvement of particular differently expressed genes in the process of PPV infection is discussed.

  8. Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Kathleen H Rubins

    Full Text Available Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV, an emerging human pathogen, and Vaccinia virus (VAC, a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA, or poly (I-C was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

  9. Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

    Science.gov (United States)

    Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

    2014-05-01

    To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. © 2013 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  10. Increased transgene expression level of rabies virus vector for transsynaptic tracing.

    Directory of Open Access Journals (Sweden)

    Shinya Ohara

    Full Text Available Viral vectors that can infect neurons transsynaptically and can strongly express foreign genes are useful for investigating the organization of neural circuits. We previously developed a propagation-competent rabies virus (RV vector based on a highly attenuated HEP-Flury strain (rHEP5.0-CVSG, which selectively infects neurons and propagates between synaptically connected neurons in a retrograde direction. Its relatively low level of transgene expression, however, makes immunostaining necessary to visualize the morphological features of infected neurons. To increase the transgene expression level of this RV vector, in this study we focused on two viral proteins: the large protein (L and matrix protein (M. We first attempted to enhance the expression of L, which is a viral RNA polymerase, by deleting the extra transcription unit and shortening the intergenic region between the G and L genes. This viral vector (rHEP5.0-GctL showed increased transgene expression level with efficient transsynaptic transport. We next constructed an RV vector with a rearranged gene order (rHEP5.0-GML with the aim to suppress the expression of M, which plays a regulatory role in virus RNA synthesis. Although this vector showed high transgene expression level, the efficiency of transsynaptic transport was low. To further evaluate the usability of rHEP5.0-GctL as a transsynaptic tracer, we inserted a fluorescent timer as a transgene, which changes the color of its fluorescence from blue to red over time. This viral vector enabled us the differentiation of primary infected neurons from secondary infected neurons in terms of the fluorescence wavelength. We expect this propagation-competent RV vector to be useful for elucidating the complex organization of the central nervous system.

  11. [Construction and identification of recombinant avian adeno-associated virus expressing GFP reporter gene].

    Science.gov (United States)

    Wang, An-ping; Sun, Huai-chang; Wang, Jian-ye; Wang, Yong-juan; Yuan, Wei-feng

    2007-07-01

    To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.

  12. Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression.

    Directory of Open Access Journals (Sweden)

    Emily A Gomme

    Full Text Available Rabies virus (RABV is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent "mark" on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre to switch neurons constitutively expressing tdTomato (red to expression of a Cre-inducible EGFP (green, permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these "cured" neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal

  13. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Science.gov (United States)

    Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

    2016-01-01

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

  14. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Directory of Open Access Journals (Sweden)

    Katherine D. Shives

    2016-10-01

    Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  15. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

    Science.gov (United States)

    Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

    2016-10-18

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5' cap with 2'-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  16. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    Science.gov (United States)

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  17. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    Science.gov (United States)

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  18. Proteomic Analysis of Pichindé virus Infection Identifies Differential Expression of Prothymosin-α

    Directory of Open Access Journals (Sweden)

    Gavin C. Bowick

    2010-01-01

    Full Text Available The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-α. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.

  19. A replication-competent foot-and-mouth disease virus expressing a luciferase reporter.

    Science.gov (United States)

    Zhang, Fuquan; Perez-Martin, Eva; Juleff, Nick; Charleston, Bryan; Seago, Julian

    2017-09-01

    Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Effect of 1918 PB1-F2 expression on influenza A virus infection kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Ribeiro, Ruy [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Smith, Amber M [UNIV OF UTAH; Adler, Frederick R [UNIV OF UTAH; Mcauley, Julie L [ST. JUDES CHILDREN RESEARCH; Mccullers, Jonathan A [ST. JUDES CHILDREN RESEARCH

    2009-01-01

    Relatively little is known about the viral factors contributing to the lethality of the 1918 pandemic, although its unparalleled virulence was likely due in part to the newly discovered PB1-F2 protein. This protein, while unnecessary for replication, increases apoptosis in monocytes, alters viral polymerase activity in vitro, and produces enhanced inflammation and increased secondary pneumonia in vivo. However, the effects the PB1-F2 protein have in vivo remain unclear. To address the mechanisms involved, we intranasally infected groups of mice with either influenza A virus PR8 or a genetically engineered virus that expresses the 1918 PB1-F2 protein on a PR8 background, PR8-PB1-F2(1918). Mice inoculated with PR8 had viral concentrations peaking at 72 hours, while those infected with PR8-PB1-F2(1918) reached peak concentrations earlier, 48 hours. Mice given PR8-PB1-F2(1918) also showed a faster decline in viral loads. We fit a mathematical model to these data to estimate parameter values and select the best model. This model supports a lower viral clearance rate and higher infected cell death rate with the PR8-PB1-F2(1918) virus, although the viral production rate may also be higher. We hypothesize that the higher PR8-PB1-F2(1918) viral titers early in an infection are due to both an increase in viral production with decreased viral clearance, and that the faster decline in the later stages of infection result from elevated cell death rates. We discuss the implications these mechanisms have during an infection with a virus expressing a virulent PBI-F2 on the possibility of a pandemic and on the importance of antiviral treatments.

  1. Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection.

    Science.gov (United States)

    Wang, Jianmiao; Li, Qinghai; Xie, Jungang; Xu, Yongjian

    2015-03-14

    It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection. Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured. CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine. Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

  2. Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ

    Energy Technology Data Exchange (ETDEWEB)

    Barkhouse, Darryll A. [Department of Cancer Biology, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Faber, Milosz [Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Department of Microbiology and Immunology 1020 Locust St., Jefferson Alumni Hall, Room 465, Philadelphia, PA 19107 (United States); Hooper, D. Craig, E-mail: douglas.hooper@jefferson.edu [Department of Cancer Biology, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Department of Neurological Surgery, 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States); Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107 (United States)

    2015-01-01

    Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. - Highlights: • IFNγ expression improves attenuated rabies virus safety and immunogenicity. • IFNγ expression is safer and more immunogenic than doubling glycoprotein expression. • Co-infection with IFNγ-expressing RABV prevents wild-type rabies virus lethality. • Vaccine safety and efficacy is additive for IFNγ and double glycoprotein expression.

  3. Mechanisms of foot-and-mouth disease virus tropism inferred from differential tissue gene expression.

    Directory of Open Access Journals (Sweden)

    James J Zhu

    Full Text Available Foot-and-mouth disease virus (FMDV targets specific tissues for primary infection, secondary high-titer replication (e.g. foot and mouth where it causes typical vesicular lesions and long-term persistence at some primary replication sites. Although integrin αVβ6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVβ6 heterodimeric receptor (FMDV receptor, fibronectin (ligand of the receptor, IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1 FMDV receptor availability and accessibility, (2 type I interferon-inducible immune response, and (3 ability to clear virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions.

  4. Changes in the gene expression profile of Arabidopsis thaliana after infection with Tobacco etch virus

    Directory of Open Access Journals (Sweden)

    Jaramillo Alfonso

    2008-08-01

    Full Text Available Abstract Background Tobacco etch potyvirus (TEV has been extensively used as model system for the study of positive-sense RNA virus infecting plants. TEV ability to infect Arabidopsis thaliana varies among ecotypes. In this study, changes in gene expression of A. thaliana ecotype Ler infected with TEV have been explored using long-oligonucleotide arrays. A. thaliana Ler is a susceptible host that allows systemic movement, although the viral load is low and syndrome induced ranges from asymptomatic to mild. Gene expression profiles were monitored in whole plants 21 days post-inoculation (dpi. Microarrays contained 26,173 protein-coding genes and 87 miRNAs. Results Expression analysis identified 1727 genes that displayed significant and consistent changes in expression levels either up or down, in infected plants. Identified TEV-responsive genes encode a diverse array of functional categories that include responses to biotic (such as the systemic acquired resistance pathway and hypersensitive responses and abiotic stresses (droughtness, salinity, temperature, and wounding. The expression of many different transcription factors was also significantly affected, including members of the R2R3-MYB family and ABA-inducible TFs. In concordance with several other plant and animal viruses, the expression of heat-shock proteins (HSP was also increased. Finally, we have associated functional GO categories with KEGG biochemical pathways, and found that many of the altered biological functions are controlled by changes in basal metabolism. Conclusion TEV infection significantly impacts a wide array of cellular processes, in particular, stress-response pathways, including the systemic acquired resistance and hypersensitive responses. However, many of the observed alterations may represent a global response to viral infection rather than being specific of TEV.

  5. Bovine parainfluenza virus type 3 (PIV3) expressing the respiratory syncytial virus (RSV) attachment and fusion proteins protects hamsters from challenge with human PIV3 and RSV.

    Science.gov (United States)

    Haller, Aurelia A; Mitiku, Misrach; MacPhail, Mia

    2003-08-01

    Parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV) are the main causes of ubiquitous acute respiratory diseases of infancy and early childhood, causing 20-25 % of pneumonia and 45-50 % of bronchiolitis in hospitalized children. The primary goal of this study was to create an effective and safe RSV vaccine based on utilizing attenuated bovine PIV3 (bPIV3) as a virus vector backbone. bPIV3 had been evaluated in human clinical trials and was shown to be attenuated and immunogenic in children as young as 2 months of age. The ability of bPIV3 to function as a virus vaccine vector was explored further by introducing the RSV attachment (G) and fusion (F) genes into the bPIV3 RNA genome. The resulting virus, bPIV3/RSV(I), contained an insert of 2900 nt, comprising two translationally competent transcription units. Despite this increase in genetic material, the virus replicated to high titres in Vero cells. This recombinant virus expressed the RSV G and F proteins sufficiently to evoke a protective immune response in hamsters upon challenge with RSV or human PIV3 and to elicit RSV neutralizing and PIV3 haemagglutinin inhibition serum antibodies. In effect, a bivalent vaccine was produced that could protect vaccinees from RSV as well as PIV3. Such a vaccine would vastly reduce the respiratory disease burden, the associated hospitalization costs and, most importantly, decrease morbidity and mortality of infants, immunocompromised individuals and the elderly.

  6. Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian ‘avian-like’ H1N1 swine viruses in mice

    Science.gov (United States)

    Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

    2014-01-01

    Objectives To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian “avian-like” (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Design Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Sample Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Setting Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Main outcome measures Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Results and Conclusions Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. PMID:24373385

  7. Mature human odontoblasts express virus-recognizing toll-like receptors.

    Science.gov (United States)

    Pääkkönen, V; Rusanen, P; Hagström, J; Tjäderhane, L

    2014-10-01

    To study the expression of toll-like receptors (TLR) -3, -7, -8 and -9 as well as interferon receptors alpha and gamma (IFNAR1/IFNAR2 and IFNGR1/IFNGR2), which play important roles in the defence against viruses. DNA microarray and quantitative PCR analyses of TLR3, -7, -8 and -9 as well as IFNAR1/IFNAR2 and IFNGR1/IFNGR2 genes in mature native human odontoblasts and pulp were performed. Immunohistochemistry was used to confirm TLR8 protein in odontoblasts of healthy and carious human teeth. TLR3, -7, -8 and -9 mRNAs were detected both in odontoblasts and in pulp, but TLR8 expression level was higher in the odontoblasts. IFNAR and IFNGR expression was observed in both tissues. Immunohistochemical analysis of healthy teeth revealed positive TLR8 staining in the pre-dentine and the dentine but varying staining patterns in the different portions of tooth. Lighter TLR8 staining was observed in dentine of mildly carious teeth. In teeth with carious lesions extending into the mid-dentine, only very weak staining was detected. The finding of these virus-recognition-related genes in odontoblasts strengthens the view that odontoblasts participate in the immune response of the dentine-pulp complex. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  8. Stable human lymphoblastoid cell lines constitutively expressing hepatitis C virus proteins.

    Science.gov (United States)

    Wölk, Benno; Gremion, Christel; Ivashkina, Natalia; Engler, Olivier B; Grabscheid, Benno; Bieck, Elke; Blum, Hubert E; Cerny, Andreas; Moradpour, Darius

    2005-06-01

    The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoid x T hybrid cell lines constitutively expressing either the NS3-4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3-4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.

  9. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

    Science.gov (United States)

    Ricklin, Meret E; Vielle, Nathalie J; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4(+) T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

  10. Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Backovic Ana

    2012-09-01

    Full Text Available Abstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus. We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

  11. The Effect of West Nile Virus Infection on the Midgut Gene Expression of Culex pipiens quinquefasciatus Say (Diptera: Culicidae).

    Science.gov (United States)

    Smartt, Chelsea T; Shin, Dongyoung; Anderson, Sheri L

    2016-12-19

    The interaction of the mosquito and the invading virus is complex and can result in physiological and gene expression alterations in the insect. The association of West Nile virus (WNV) and Culex pipiens quinquefasciatus mosquitoes results in measurable changes in gene expression; 22 gene products were shown previously to have altered expression. Sequence analysis of one product, CQ G1A1, revealed 100% amino acid identity to gram negative bacteria binding proteins (CPQGBP) in Cx. p. quinquefasciatus, Aedes aegypti (70%) and Anopheles gambiae (63%) that function in pathogen recognition. CQ G1A1 also was differentially expressed following WNV infection in two populations of Cx. p. quinquefasciatus colonized from Florida with known differences in vector competence for WNV and showed spatial and temporal gene expression differences in midgut, thorax, and carcass tissues. These data suggest gene expression of CQ G1A1 is influenced by WNV infection and the WNV infection-controlled expression differs between populations and tissues.

  12. Cloning and expression of human papilloma virus type 6b-L1 gene

    Directory of Open Access Journals (Sweden)

    Lie-hua DENG

    2011-03-01

    Full Text Available Objective To investigate the expression of green fluorescent protein plasmid of human papilloma virus 6b L1 gene(HPV6bL1 in eukaryotic cells.Methods The L1 gene of PQE40-HPV6bL1 was amplified by PCR,purified by restriction enzyme digestion,and then connected to eukaryotic expression plasmid PEGFP-C1.The recombinant expression vector was then transformed into E.coli DH5a,which was identified by BamH Ⅰ and Hand Ⅲ digestion and the positive vector was selected.The recombinant plasmid PEGFP-HPV6bL1 was transfected into COS-7 cells by liposomal transfection technique and the expression of fusion protein was observed under fluorescence microscope.The generation of HPV6bL1 mRNA was detected by RT-PCR.Results Identification of PEGFP-HPV6bL1 by enzyme digestion and sequencing showed that the length,direction and inserted location of target,which was inserted into the recombinant,was correct and the expression of EGFP in transfected cell was observed.Conclusions A new type of green fluorescent HPV6bL1 eukaryotic expression system has been established.It may provide a research foundation for the study of the protein.

  13. Assembly of SIV virus-like particles containing envelope proteins using a baculovirus expression system.

    Science.gov (United States)

    Yamshchikov, G V; Ritter, G D; Vey, M; Compans, R W

    1995-12-01

    The requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gag precursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envt protein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP.

  14. Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Guillermo, Leon M; Picton, D D; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2013-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Regulation of core expression during the hepatitis C virus life cycle.

    Science.gov (United States)

    Afzal, Muhammad Sohail; Alsaleh, Khaled; Farhat, Rayan; Belouzard, Sandrine; Danneels, Adeline; Descamps, Véronique; Duverlie, Gilles; Wychowski, Czeslaw; Zaidi, Najam us Sahar Sadaf; Dubuisson, Jean; Rouillé, Yves

    2015-02-01

    Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle. © 2015 The Authors.

  16. Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed.

    Directory of Open Access Journals (Sweden)

    Xuezhong Cai

    2006-03-01

    Full Text Available The pathogenic lymphocryptovirus Epstein-Barr virus (EBV is shown to express at least 17 distinct microRNAs (miRNAs in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.

  17. RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression

    OpenAIRE

    Sanna M Mäkelä; Österlund, Pamela; Westenius, Veera; Latvala, Sinikka; Diamond, Michael S.; Gale, Michael; Julkunen, Ilkka

    2015-01-01

    Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechan...

  18. Recombinant capripoxviruses expressing proteins of bluetongue virus: evaluation of immune responses and protection in small ruminants.

    Science.gov (United States)

    Perrin, Aurélie; Albina, Emmanuel; Bréard, Emmanuel; Sailleau, Corinne; Promé, Sylvie; Grillet, Colette; Kwiatek, Olivier; Russo, Pierre; Thiéry, Richard; Zientara, Stephan; Cêtre-Sossah, Catherine

    2007-09-17

    The development of recombinant capripoxviruses for protective immunization of ruminants against bluetongue virus (BTV) infection is described. Sheep (n=11) and goats (n=4) were immunized with BTV recombinant capripoxviruses (BTV-Cpox) individually expressing four different genes encoding two capsid proteins (VP2 and VP7) and two non-structural proteins (NS1, NS3) of BTV serotype 2 (BTV-2). Seroconversion was observed against NS3, VP7 and VP2 in both species and a lymphoproliferation specific to BTV antigens was also demonstrated in goats. Finally, partial protection of sheep challenged 3 weeks after BTV-Cpox administration with a virulent strain of BTV-2, was observed.

  19. Vesicular Stomatitis Virus Infection Promotes Immune Evasion by Preventing NKG2D-Ligand Surface Expression

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Nielsen, Jens

    2011-01-01

    Vesicular stomatitis virus (VSV) has recently gained attention for its oncolytic ability in cancer treatment. Initially, we hypothesized that VSV infection could increase immune recognition of cancer cells through induction of the immune stimulatory NKG2D-ligands. Here we show that VSV infection......D-ligand expression at an early post-transcriptional level. Our results show that VSV possess an escape mechanism, which could affect the immune recognition of VSV infected cancer cells. This may also have implications for immune recognition of cancer cells after combined treatment with VSV...

  20. Expression and Functional Characterization of Bluetongue Virus VP2 Protein: Role in Cell Entry

    OpenAIRE

    Hassan, Sharifah S; Roy, Polly

    1999-01-01

    Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determ...

  1. Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

    NARCIS (Netherlands)

    E.J. Tijhaar (Edwin); C.H.J. Siebelink (Kees); J.A. Karlas (Jos); M.C. Burger; F.R. Mooi (Frits); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractSalmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens

  2. Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis

    Directory of Open Access Journals (Sweden)

    Skjaeret Camilla

    2006-12-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease. Results Among the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27 and the highly downregulated gene Charcot-Leyden crystal protein (CLC to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray. Conclusion The gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

  3. Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

    Directory of Open Access Journals (Sweden)

    Okagawa Tomohiro

    2012-05-01

    Full Text Available Abstract The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3 and its ligand, galectin-9 (Gal-9, are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.

  4. Structure and Immunogenicity of Alternative Forms of the Simian Immunodeficiency Virus Gag Protein Expressed Using Venezuelan Equine Encephalitis Virus Replicon Particles

    OpenAIRE

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T.; Swanstrom, Ronald; Davis, Nancy L.

    2007-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infect...

  5. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

  6. Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species.

    Science.gov (United States)

    Martins, Mathias; Joshi, Lok R; Rodrigues, Fernando S; Anziliero, Deniz; Frandoloso, Rafael; Kutish, Gerald F; Rock, Daniel L; Weiblen, Rudi; Flores, Eduardo F; Diel, Diego G

    2017-11-01

    The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV∆024RABV-G or ORFV∆121RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV∆024RABV-G and ORFV∆121RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV∆121RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV∆024RABV-G-immunized animals, indicating a higher immunogenicity of ORFVΔ121-based vectors in these animal species. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

    Science.gov (United States)

    Huai, L; Chiocca, S M; Gilbreth, M A; Ainsworth, J R; Bishop, L A; Murphy, E C

    1992-09-01

    We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.

  8. Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

    Science.gov (United States)

    Tsujimoto, Hitoshi; Hanley, Kathryn A; Sundararajan, Anitha; Devitt, Nicholas P; Schilkey, Faye D; Hansen, Immo A

    2017-01-01

    The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

  9. Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Hitoshi Tsujimoto

    Full Text Available The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal.We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses.Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

  10. Restricted expression of Borna disease virus glycoprotein in brains of experimentally infected Lewis rats.

    Science.gov (United States)

    Werner-Keiss, N; Garten, W; Richt, J A; Porombka, D; Algermissen, D; Herzog, S; Baumgärtner, W; Herden, C

    2008-12-01

    Borna disease virus (BDV) induces a persistent infection in the central nervous system (CNS) accompanied by a non-purulent meningoencephalitis. BDV-infection of Lewis rats provides an important model to investigate basic principles of neurotropism, viral persistence and resulting dysfunctions. To date, the in vivo strategies of BDV to persist in the CNS are not fully understood. Viral glycoproteins are main targets of the antiviral defence implicating a controlled expression in case of persistent infections. Therefore, we analysed the expression profiles of the BDV-glycoprotein (BDV-GP) and corresponding BDV-intron II RNA in experimentally infected rat brains, focusing on their spatio-temporal occurrence, regional, cellular and intracellular locations. This was carried out by immunohistochemistry and in situ hybridization. The expression pattern of the most abundantly expressed BDV-nucleoprotein (BDV-N) served as a reference. BDV-N mRNA was detected preferentially in the cytoplasm of neurones, whereas BDV-intron II mRNA was found predominantly in the nucleus of brain cells. The genomic RNA was restricted to the nucleus. Expression of BDV-GP was significantly lower than BDV-N expression and mainly limited to cerebral cortex, hippocampus, amygdala and thalamus. BDV-GP was restricted to larger neurones; BDV-N occurred also in astrocytes, oligodendrocytes and ependymal cells. The expression profiles of BDV-GP, BDV-N and their mRNAs are significantly different, indicating that BDV-GP expression is regulated in vivo. This might be achieved by restricted nuclear export and/or maturation of BDV-intron II mRNA or limited translation as a viral mechanism to escape from the immune response and enable persistence in the CNS.

  11. Regulation of gene expression in adeno-associated virus vectors in the brain.

    Science.gov (United States)

    Haberman, Rebecca P; McCown, Thomas J

    2002-10-01

    Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application. Copyright 2002 Elsevier Science (USA)

  12. Gene expression of the TGF-beta family in rat brain infected with Borna disease virus.

    Science.gov (United States)

    Nishino, Yoshii; Ooishi, Ryo; Kurokawa, Sachiko; Fujino, Kan; Murakami, Masaru; Madarame, Hiroo; Hashimoto, Osamu; Sugiyama, Kazutoshi; Funaba, Masayuki

    2009-01-01

    CRNP5, a variant of Borna disease virus (BDV), has stronger pathogenesis in rats than the related variant CRP3, although only 4 amino acids in the whole genome are different. As a first step to clarify the differential pathogenesis between the variants, the present study focused on examining the expression of the transforming growth factor (TGF)-beta family in the brain of rats infected with BDV. The main results were as follows. (1) BDV infection, irrespective of the variant, up-regulates TGF-beta1 expression in the brain, (2) the expressions of signal receptors for TGF-beta1 are also increased, (3) the expression of brain inhibin/activin betaE is up-regulated by BDV infection, and (4) the expression of brain inhibin/activin betaC tends to be higher in rats exhibiting severe Borna disease. These results indicate that members of the TGF-beta family are involved in neuronal disorders induced by BDV infection in a ligand-dependent manner. In particular, up-regulation of inhibin/activin betaC may be a key event responsible for induction of the stronger pathogenesis of the CRNP5 variant of BDV.

  13. Human Papilloma Virus-Dependent HMGA1 Expression Is a Relevant Step in Cervical Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Massimiliano Mellone

    2008-08-01

    Full Text Available HMGA1 is a member of a small family of architectural transcription factors involved in the coordinate assembly of multiprotein complexes referred to as enhanceosomes. In addition to their role in cell proliferation, differentiation, and development, high-mobility group proteins of the A type (HMGA family members behave as transforming protoncogenes either in vitro or in animal models. Recent reports indicated that HMGA1 might counteract p53 pathway and provided an interesting hint on the mechanisms determining HMGA's transforming potential. HMGA1 expression is deregulated in a very large array of human tumors, including cervical cancer, but very limited information is available on the molecular mechanisms leading to HMGA1 deregulation in cancer cells. Here, we report that HMGA1 expression is sustained by human papilloma virus (HPV E6/E7 proteins in cervical cancer, as demonstrated by either E6/E7 overexpression or by repression through RNA interference. Knocking down HMGA1 expression by means of RNA interference, we also showed that it is involved in cell proliferation and contributes to p53 inactivation in this type of neoplasia. Finally, we show that HMGA1 is necessary for the full expression of HPV18 E6 and E7 oncoproteins thus establishing a positive autoregulatory loop between HPV E6/E7 and HMGA1 expression.

  14. PCR array analysis of gene expression profiles in chronic active Epstein-Barr virus infection.

    Science.gov (United States)

    Murakami, Masanao; Hashida, Yumiko; Imajoh, Masayuki; Maeda, Akihiko; Kamioka, Mikio; Senda, Yasutaka; Sato, Tetsuya; Fujieda, Mikiya; Wakiguchi, Hiroshi; Daibata, Masanori

    2014-07-01

    To determine the host cellular gene expression profiles in chronic active Epstein-Barr virus infection (CAEBV), peripheral blood samples were obtained from three patients with CAEBV and investigated using a PCR array analysis that focused on T-cell/B-cell activation. We identified six genes with expression levels that were tenfold higher in CAEBV patients compared with those in healthy controls. These results were verified by quantitative reverse transcription-PCR. We identified four highly upregulated genes, i.e., IL-10, IL-2, IFNGR1, and INHBA. These genes may be involved in inflammatory responses and cell proliferation, and they may contribute to the development and progression of CAEBV. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. Expression and purification of human papillomavirus 18 L1 virus-like particle from saccharomyces cerevisiae.

    Science.gov (United States)

    Woo, Mi-Kyung; An, Jung-Mo; Kim, Jun-Dong; Park, Sue-Nie; Kim, Hong-Jin

    2008-02-01

    Cervical cancer caused by human papillomavirus (HPV) might be successfully prevented by HPV vaccination and screening. HPV vaccination and HPV serology assays have been investigated using HPV virus-like particles (VLPs). In this study we produced HPV18 L1 VLPs in Saccharomyces cerevisiae and purified them. The HPV18 L1 gene was cloned into the yeast expression vector YEGalpha-HIR525, and transformed into Saccharomyces cerevisiae. Expression of HPV18 L1 protein was demonstrated by Western blotting. The HPV18 L1 protein was purified by ultracentrifugation, size-exclusion chromatography and cation-exchange chromatography, and was up to 95% pure. We showed by transmission electron microscopy that the purified protein self-assembled into VLPs. These findings should be useful for establishing vaccine efficacy as well as characterizing vaccine candidates, and may provide an international reference standard for HPV serology assays.

  16. Phenotypic and Transcriptomic Analysis of Nicotiana benthamiana Expressing Cucumber mosaic virus 2b gene

    Directory of Open Access Journals (Sweden)

    Seong-Han Sohn

    2015-09-01

    Full Text Available Cucumber mosaic virus possesses 2b gene known as a suppressor of post-transcriptional gene silencing (PTGS. To investigate its function and effect in plant, transgenic Nicotiana benethamiana expressing 2b gene was developed and analyzed in phenotypic characteristics and differential gene expression (DEG comparing with wild-type. Eight lines of transgenic plants (T0 were obtained with difficulty and showed severe deformed phenotypes in leaves, flowers, petioles and etc. Moreover, transgenic plants were hardly able to set seeds, but small amounts of seeds were barely produced in some of transgene-hemizygous plants. DEG analysis showed that transgenic plant ectopically accumulated diverse RNA transcripts at higher levels than wild-type probably due to the disturbance in RNA metabolism, especially of RNA decay, caused by 2b-mediated inhibition of PTGS. These ectopic accumulations of RNAs disrupt protein and RNA homeostasis and then subsequently lead to abnormal phenotypes of transgenic plants.

  17. Expression of CD154 by a Simian Immunodeficiency Virus Vector Induces Only Transitory Changes in Rhesus Macaques

    OpenAIRE

    Vida L. Hodara; Velasquillo, M. Cristina; Parodi, Laura M.; Giavedoni, Luis D.

    2005-01-01

    Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that...

  18. Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

    Science.gov (United States)

    Dinh, Phat X.; Beura, Lalit K.; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K.

    2011-01-01

    Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections. PMID:21752917

  19. Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine.

    Science.gov (United States)

    Luo, Jun; Zhang, Boyue; Wu, Yuting; Tian, Qin; Zhao, Jing; Lyu, Ziyu; Zhang, Qiong; Mei, Mingzhu; Luo, Yongwen; Guo, Xiaofeng

    2017-02-07

    Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEP-CaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8 + T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN.

    Science.gov (United States)

    Whitfield, Anna E; Ullman, Diane E; German, Thomas L

    2004-12-01

    Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.

  1. Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

    Science.gov (United States)

    Desuzinges Mandon, Elodie; Traversier, Aurélien; Champagne, Anne; Benier, Lorraine; Audebert, Stéphane; Balme, Sébastien; Dejean, Emmanuel; Rosa Calatrava, Manuel; Jawhari, Anass

    2017-03-01

    Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression.

    Directory of Open Access Journals (Sweden)

    Jason Lamontagne

    2016-02-01

    Full Text Available Globally, a chronic hepatitis B virus (HBV infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.

  3. Monocytes inhibit hepatitis C virus-induced TRAIL expression on CD56bright NK cells.

    Science.gov (United States)

    Mele, Dalila; Mantovani, Stefania; Oliviero, Barbara; Grossi, Giulia; Lombardi, Andrea; Mondelli, Mario U; Varchetta, Stefania

    2017-12-01

    Natural killer (NK) cells play an important role in the pathogenesis of hepatitis C virus (HCV) infection. We have previously shown that culture-derived HCV (HCVcc) enhance tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) expression on healthy NK cells, but not on those from patients infected with HCV, which was likely dependent on accessory cells. Here we sought to elucidate the mechanisms involved in altered TRAIL upregulation in this setting. Peripheral blood mononuclear cells (PBMC) from controls and patients infected with HCV were exposed to HCVcc. Cell depletions were performed to identify cells responsible for NK cell activation. Flow cytometry and ELISA were used to identify the cytokines involved in the NK activation process. In patients infected with HCV, soluble factors secreted by control PBMC restored the ability of NK cells to express TRAIL. Of note, CD14+ cell depletion had identical effects upon virus exposure and promoted increased degranulation. Moreover, increased concentrations of interleukin (IL)-18 binding protein a (IL-18BPa) and IL-36 receptor antagonist (IL-36RA) were observed after PBMC exposure to HCVcc in patients with HCV. HCVcc-induced NK cell TRAIL expression was inhibited by IL-18BPa and IL-36RA in control subjects. There were statistically significant correlations between IL-18BPa and indices of liver inflammation and fibrosis, supporting a role for this protein in the pathogenesis of chronic HCV infection. During chronic HCV infection, monocytes play a key role in negative regulation of NK cell activation, predominantly via secretion of inhibitors of IL-18 and IL-36. Coordination and collaboration between immune cells are essential to fight pathogens. Herein we show that during HCV infection monocytes secrete IL-18 and IL-36 inhibitory proteins, reducing NK cell activation, and consequently inhibiting their ability to express TRAIL and kill target cells. Copyright © 2017 European Association for the Study of the

  4. Alpha-mangostin inhibits both dengue virus production and cytokine/chemokine expression.

    Science.gov (United States)

    Tarasuk, Mayuri; Songprakhon, Pucharee; Chimma, Pattamawan; Sratongno, Panudda; Na-Bangchang, Kesara; Yenchitsomanus, Pa-Thai

    2017-08-15

    Since severe dengue virus (DENV) infection in humans associates with both high viral load and massive cytokine production - referred to as "cytokine storm", an ideal drug for treatment of DENV infection should efficiently inhibit both virus production and cytokine expression. In searching for such an ideal drug, we discovered that α-mangostin (α-MG), a major bioactive compound purified from the pericarp of the mangosteen fruit (Garcinia mangostana Linn), which has been used in traditional medicine for several conditions including trauma, diarrhea, wound infection, pain, fever, and convulsion, inhibits both DENV production in cultured hepatocellular carcinoma HepG2 and Huh-7 cells, and cytokine/chemokine expression in HepG2 cells. α-MG could also efficiently inhibit all four serotypes of DENV. Treatment of DENV-infected cells with α-MG (20μM) significantly reduced the infection rates of four DENV serotypes by 47-55%. α-MG completely inhibited production of DENV-1 and DENV-3, and markedly reduced production of DENV-2 and DENV-4 by 100 folds. Furthermore, it could markedly reduce cytokine (IL-6 and TNF-α) and chemokine (RANTES, MIP-1β, and IP-10) transcription. These actions of α-MG are more potent than those of antiviral agent (ribavirin) and anti-inflammatory drug (dexamethasone). Thus, α-MG is potential to be further developed as therapeutic agent for DENV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Improved microarray gene expression profiling of virus-infected cells after removal of viral RNA

    Directory of Open Access Journals (Sweden)

    Rottier Peter JM

    2008-05-01

    Full Text Available Abstract Background Sensitivity and accuracy are key points when using microarrays to detect alterations in gene expression under different conditions. Critical to the acquisition of reliable results is the preparation of the RNA. In the field of virology, when analyzing the host cell's reaction to infection, the often high representation of viral RNA (vRNA within total RNA preparations from infected cells is likely to interfere with microarray analysis. Yet, this effect has not been investigated despite the many reports that describe gene expression profiling of virus-infected cells using microarrays. Results In this study we used coronaviruses as a model to show that vRNA indeed interferes with microarray analysis, decreasing both sensitivity and accuracy. We also demonstrate that the removal of vRNA from total RNA samples, by means of virus-specific oligonucleotide capturing, significantly reduced the number of false-positive hits and increased the sensitivity of the method as tested on different array platforms. Conclusion We therefore recommend the specific removal of vRNA, or of any other abundant 'contaminating' RNAs, from total RNA samples to improve the quality and reliability of microarray analyses.

  6. Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.

    OpenAIRE

    Caley, I J; Betts, M R; Irlbeck, D M; Davis, N L; Swanstrom, R; Frelinger, J A; Johnston, R E

    1997-01-01

    A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. T...

  7. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    Science.gov (United States)

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-02-19

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

  8. Expression of the Epstein-Barr virus gp350/220 gene in rodent and primate cells.

    Science.gov (United States)

    Whang, Y; Silberklang, M; Morgan, A; Munshi, S; Lenny, A B; Ellis, R W; Kieff, E

    1987-06-01

    The gene encoding the Epstein-Barr virus envelope glycoproteins gp350 and gp220 was inserted downstream of the cytomegalovirus immediate-early, Moloney murine leukemia virus, mouse mammary tumor virus, or varicella-zoster virus gpI promoters in vectors containing selectable markers. Host cell and recombinant vector systems were defined which enabled the isolation of rodent or primate cell clones which expressed gp350/220 in substantial quantities. Continued expression of gp350/220 required maintenance of cells under positive selection for linked markers and periodic cloning. gp350/220 expressed in various host cells varied slightly in electrophoretic mobility, probably reflecting differences in glycosylation. Insertion of a stop codon into the gp350/220 open reading frame, upstream of the putative membrane anchor sequence, resulted in efficient secretion of truncated gp350 and gp220 from rat pituitary (GH3) cells. gp350/220 expressed in mammalian cells is highly immunogenic and elicits virus-neutralizing antibodies when administered to mice.

  9. Exploiting plant virus-derived components to achieve in planta expression and for templates for synthetic biology applications.

    Science.gov (United States)

    Saunders, Keith; Lomonossoff, George P

    2013-10-01

    This review discusses the varying roles that have been played by many plant-viral regulatory sequences and proteins in the creation of plant-based expression systems and virus particles for use in nanotechnology. Essentially, there are two ways of expressing an exogenous protein: the creation of transgenic plants possessing a stably integrated gene construction, or the transient expression of the desired gene following the infiltration of the gene construct. Both depend on disarmed strains of Agrobacterium tumefaciens to deliver the created gene construction into cell nuclei, usually through the deployment of virus-derived components. The importance of efficient mRNA translation in the latter process is highlighted. Plant viruses replicate to sustain an infection to promote their survival. The major product of this, the virus particle, is finding increasing roles in the emerging field of bionanotechnology. One of the major products of plant-viral expression is the virus-like particle (VLP). These are increasingly playing a role in vaccine development. Similarly, many VLPs are suitable for the investigation of the many facets of the emerging field of synthetic biology, which encompasses the design and construction of new biological functions and systems not found in nature. Genetic and chemical modifications to plant-generated VLPs serve as ideal starter templates for many downstream synthetic biology applications. © 2013 JIC. New Phytologist © 2013 New Phytologist Trust.

  10. The Expression of the Hepatocyte SLAMF3 (CD229) Receptor Enhances the Hepatitis C Virus Infection

    Science.gov (United States)

    Cartier, Flora; Marcq, Ingrid; Douam, Florian; Ossart, Christèle; Regnier, Aline; Debuysscher, Véronique; Lavillette, Dimitri; Bouhlal, Hicham

    2014-01-01

    Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes. PMID:24927415

  11. Effect of human papilloma virus expression on clinical course of laryngeal papilloma.

    Science.gov (United States)

    Kim, Kwang Moon; Cho, Nam Hoon; Choi, Hong Shik; Kim, Young Ho; Byeon, Hyung Kwon; Min, Hyun Jin; Kim, Se-Heon

    2008-10-01

    Our observations suggest that human papilloma virus (HPV) 6/11 is the main causative agent of laryngeal papilloma and that detection of active HPV DNA expression may be helpful in identifying patients with aggressive recurrent laryngeal papilloma. HPV is assumed to be the main causative agent of this disease. We investigated the expression of the entire genotype of HPV in cases of laryngeal papilloma and correlated their expression with the clinical course of the disease. Seventy cases of laryngeal papilloma were evaluated for the presence of the HPV genome by in situ hybridization (ISH) using wide-spectrum HPV DNA probe. Specific types of HPV infection were determined by DNA ISH using type-specific HPV DNA probes (HPV 6, 11, 16, 18, 31, 33). Separate analyses were conducted comparing viral types, frequency of recurrences and duration of disease-free periods. We detected HPV DNA in 40 of the 70 laryngeal papilloma cases (57%). In particular, HPV DNA was detected in 75% of the juvenile types. There were significant associations between HPV and laryngeal papilloma (p<0.01). Among the HPV-positive cases, major specific types were HPV 6/11 (97%). Significant associations were also noted between viral expression and clinical course.

  12. The expression of the hepatocyte SLAMF3 (CD229 receptor enhances the hepatitis C virus infection.

    Directory of Open Access Journals (Sweden)

    Flora Cartier

    Full Text Available Hepatitis C virus (HCV is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3 in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

  13. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    Science.gov (United States)

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  14. A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

    Science.gov (United States)

    Ryder, Alex B.; Buonocore, Linda; Vogel, Leatrice; Nachbagauer, Raffael; Krammer, Florian

    2014-01-01

    ABSTRACT The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCE Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell

  15. A viable recombinant rhabdovirus lacking its glycoprotein gene and expressing influenza virus hemagglutinin and neuraminidase is a potent influenza vaccine.

    Science.gov (United States)

    Ryder, Alex B; Buonocore, Linda; Vogel, Leatrice; Nachbagauer, Raffael; Krammer, Florian; Rose, John K

    2015-03-01

    The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a

  16. Isolation and propagation of Dengue virus in Vero and BHK-21 cells expressing human DC-SIGN stably.

    Science.gov (United States)

    Phanthanawiboon, Supranee; A-nuegoonpipat, Atchareeya; Panngarm, Narawan; Limkittikul, Kriengsak; Ikuta, Kazuyoshi; Anantapreecha, Surapee; Kurosu, Takeshi

    2014-12-01

    The "standard" methods of isolating dengue virus (DENV) utilize the mosquito cell line C6/36, monkey kidney LLC-MK2 cells, Vero cells, or baby hamster kidney (BHK-21) cells. However, these cells lines lack a particular DENV receptor, known as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is expressed on immature dendritic cells and monocytes/macrophages. This may result in less efficient virus isolation and propagation. The present study used a lentivirus vector to establish Vero and BHK-21 cell lines (Vero-DC and BHK-DC) that express human DC-SIGN stably. Five DENV strains, each passaged several times in C6/36 cells, replicated more efficiently in Vero-DC and BHK-DC than in the parental Vero or BHK-21 cells. Vero/Vero-DC and BHK-21/BHK-DC were used to isolate virus from buffy coats and plasma samples derived from 13 patients infected with DENV. Most of the viruses showed increased production in cell lines expressing DC-SIGN. However, the isolation rate was lower (15.4-46.2%) than that from C6/36 cells (84.6%). Interestingly, when the viruses were isolated in C6/36 cells prior to infecting Vero/Vero-DC and BHK-21/BHK-DC, the rate of virus production increased markedly, reaching levels higher than those initially achieved in C6/36 cells. These data suggest that Vero-DC and BHK-DC could be useful tools for virus propagation, and that human specimens may contain a factor that interferes with virus growth in mammalian cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus

    Directory of Open Access Journals (Sweden)

    Vera L. Tunitskaya

    2016-10-01

    Full Text Available Hepatitis delta virus (HDV is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg, its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

  18. Gene Expression Analysis of Plum pox virus (Sharka Susceptibility/Resistance in Apricot (Prunus armeniaca L..

    Directory of Open Access Journals (Sweden)

    Manuel Rubio

    Full Text Available RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925, which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein PPVres region could also be involved in the resistance.

  19. Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

    Science.gov (United States)

    Rubio, Manuel; Ballester, Ana Rosa; Olivares, Pedro Manuel; Castro de Moura, Manuel; Dicenta, Federico; Martínez-Gómez, Pedro

    2015-01-01

    RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

  20. VIRUSES

    Indian Academy of Sciences (India)

    and-mouth disease in livestock was an infectious particle smaller than any bacteria. This was the first clue to the nature of viruses, genetic entities that lie somewhere in the gray area between living and non-living states.

  1. De novo foliar transcriptome of Chenopodium amaranticolor and analysis of its gene expression during virus-induced hypersensitive response.

    Science.gov (United States)

    Zhang, Yongqiang; Pei, Xinwu; Zhang, Chao; Lu, Zifeng; Wang, Zhixing; Jia, Shirong; Li, Weimin

    2012-01-01

    The hypersensitive response (HR) system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10(-5). Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory

  2. Abnormal CD40 Ligand (CD154) Expression in Human Immunodeficiency Virus-Infected Children

    Science.gov (United States)

    O'Gorman, Maurice R. G.; DuChateau, Brian; Paniagua, Mary; Hunt, Janelle; Bensen, Nicolas; Yogev, Ram

    2001-01-01

    The CD40 ligand (CD154), expressed primarily on activated CD4-positive T cells, is a costimulatory molecule involved in B-cell proliferation, germinal center formation, and immunoglobulin class switching. Since B-cell abnormalities including hypergammaglobulinemia and abnormal antibody-specific immune responses are prominent and occur early during the course of pediatric human immunodeficiency virus (HIV) infection, we measured the baseline levels and the induced levels of expression of CD154 on CD3+ CD8− (T helper cells) in HIV-infected children and uninfected children born to HIV-positive mothers. The percentage of CD154+ T helper cells activated in vitro and the level of CD154 expressed per T helper cell (mean fluorescent channel [MFC]) were significantly lower in the HIV-infected children than in the uninfected control group (77% ± 3% versus 89% ± 1%, respectively [P CD154 expressed on resting T helper cells in the HIV-infected group were not significantly different from the levels observed in the control group. In the HIV-infected children, the level of CD154 on activated T helper cells correlated with the level of immunodeficiency, as assessed by the CD4 T-cell levels (correlation coefficient [r] = 0.707, P = 0.003), but did not correlate with the viral load or with any of the serum immunoglobulin concentrations measured in this group of HIV-infected children. The baseline level of CD154 expressed on T helper cells did, however, correlate with the concentration of immunoglobulin A in serum. We conclude that HIV-infected children have impaired regulation of CD154 expression which may contribute to the immune dysregulation commonly observed. PMID:11687447

  3. Expression of a synthetic rust fungal virus cDNA in yeast.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; Garrett, Wesley M

    2016-01-01

    Mycoviruses are viruses that infect fungi. Recently, mycovirus-like RNAs were sequenced from the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. One of the RNAs appeared to represent a novel mycovirus and was designated Phakopsora pachyrhizi virus 2383 (PpV2383). The genome of PpV2383 resembles Saccharomyces cerevisiae virus L-A, a double-stranded (ds) RNA mycovirus of yeast. PpV2383 encodes two major, overlapping open reading frames with similarity to gag (capsid protein) and pol (RNA-dependent RNA polymerase), and a -1 ribosomal frameshift is necessary for the translation of a gag-pol fusion protein. Phylogenetic analysis of pol relates PpV2383 to members of the family Totiviridae, including L-A. Because the obligate biotrophic nature of P. pachyrhizi makes it genetically intractable for in vivo analysis and because PpV2383 is similar to L-A, we synthesized a DNA clone of PpV2383 and tested its infectivity in yeast cells. PpV2383 RNA was successfully expressed in yeast, and mass spectrometry confirmed the translation of gag and gag-pol fusion proteins. There was, however, no production of PpV2383 dsRNA, the evidence of viral replication. Neither the presence of endogenous L-A nor the substitution of the 5' and 3' untranslated regions with those from L-A was sufficient to rescue replication of PpV2383. Nevertheless, the proof of transcription and translation from the clone in vivo are steps toward confirming that PpV2383 is a mycovirus. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and such research may facilitate biological control of rust diseases.

  4. Expression and functional characterization of bluetongue virus VP2 protein: role in cell entry.

    Science.gov (United States)

    Hassan, S S; Roy, P

    1999-12-01

    Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determine various biological functions of VP2. The purified VP2 was shown to have virus hemagglutinin activity and was antigenically indistinguishable from the VP2 of the virion. Whether VP2 is responsible for BTV entry into permissive cells was subsequently assessed by cell surface attachment and internalization studies with an immunofluorescence assay system. The results demonstrated that VP2 alone is responsible for virus entry into mammalian cells. By competition assay, it appeared that both VP2 and the BTV virion attached to the same cell surface molecule(s). The purified VP2 also had a strong affinity for binding to glycophorin A, a sialoglycoprotein component of erythrocytes, indicating that VP2 may be responsible for BTV transmission by the Culicoides vector to vertebrate hosts during blood feeding. Further, by various enzymatic treatments of BTV-permissive L929 cells, preliminary data have been obtained which indicated that the BTV receptor molecule(s) is likely to be a glycoprotein and that either the protein moiety of the glycoprotein or a second protein molecule could also serve as a coreceptor for BTV infection.

  5. Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

    Directory of Open Access Journals (Sweden)

    Pinghua Li

    Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

  6. A novel borna disease virus vector system that stably expresses foreign proteins from an intercistronic noncoding region.

    Science.gov (United States)

    Daito, Takuji; Fujino, Kan; Honda, Tomoyuki; Matsumoto, Yusuke; Watanabe, Yohei; Tomonaga, Keizo

    2011-12-01

    Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5' untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions.

  7. A Novel Borna Disease Virus Vector System That Stably Expresses Foreign Proteins from an Intercistronic Noncoding Region▿

    Science.gov (United States)

    Daito, Takuji; Fujino, Kan; Honda, Tomoyuki; Matsumoto, Yusuke; Watanabe, Yohei; Tomonaga, Keizo

    2011-01-01

    Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5′ untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions. PMID:21937656

  8. Expression Profile of Interferon Regulatory Factor 1 in Chronic Hepatitis B Virus-Infected Liver Transplant Patients.

    Science.gov (United States)

    Janfeshan, Sahar; Yaghobi, Ramin; Eidi, Akram; Karimi, Mohammad Hossein; Geramizadeh, Bita; Malekhosseini, Seyed Ali; Kafilzadeh, Farshid

    2017-12-01

    Hepatitis B virus, which mainly affects normal liver function, leads to severe acute and chronic hepatitis, resulting in cirrhosis and hepatocellular carcinoma, but can be safely treated after liver transplant. Evaluation of determinative biomarkers may facilitate more effective treatment of posttransplant rejection. Therefore, we investigated interferon regulatory factor 1 expression in hepatitis B virus-infected liver transplant patients with and without previous rejection compared with controls. Hepatitis B virus-infected liver recipients were divided into those with (20 patients) and without a rejection (26 patients), confirmed by pathologic analyses in those who had a rejection. In addition, a healthy control group composed of 13 individuals was included. Expression levels of interferon regulatory factor 1 were evaluated during 3 follow-ups after transplant using an in-house comparative SYBR green real-time polymerase chain reaction method. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 16.0, IBM Corporation, Armonk, NY, USA). Modifications of interferon regulatory factor 1 gene expression levels in patient groups with and without rejection were not significant between days 1, 4, and 7 after liver transplant. Interferon regulatory factor 1 mRNA expression levels were down-regulated in patients without rejection versus patients with rejection, although not significantly at day 1 (P = .234) and day 4 (P = .302) but significantly at day 7 (P = .004) after liver transplant. Down-regulation of interferon regulatory factor 1 gene expression in hepatitis B virus patients without rejection emphasized counteraction between hepatitis B virus replication and interferon regulatory factor 1 production. On the other hand, interferon regulatory factor 1 gene overexpression in patients with rejection may result in inflammatory reactions and ischemic-reperfusion injury. Therefore, a better understanding of the association between

  9. Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system.

    Science.gov (United States)

    Goytia, Elisa; Fernández-Calvino, Lourdes; Martínez-García, Belén; López-Abella, Dionisio; López-Moya, Juan José

    2006-11-01

    Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 5' region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.

  10. Marek's Disease Virus Infection in the Brain: Virus Replication, Cellular Infiltration, and Major Histocompatibility Complex Antigen Expression

    National Research Council Canada - National Science Library

    Gimeno, I. M; Witter, R. L; Hunt, H. D; Lee, L. F; Reddy, S. M; Neumann, U

    2001-01-01

    Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype 1 MDV representing two pathotypes (v and vv...

  11. Gene expression profiles associated with lymphocystis disease virus (LCDV) in experimentally infected Senegalese sole (Solea senegalensis).

    Science.gov (United States)

    Carballo, Carlos; Castro, Dolores; Borrego, Juan J; Manchado, Manuel

    2017-07-01

    In the present study, the pathogenesis of lymphocystis disease virus (LCDV) and the immune gene expression patterns associated with this viral infection were determined in the flatfish Senegalese sole. The results indicate that LCDV spreads rapidly from the peritoneal cavity through the bloodstream to reach target organs such as kidney, gut, liver, and skin/fin. The viral load was highest in kidney and reduced progressively thorough the experiment in spite of the viral major capsid protein gene was transcribed. The LCDV injection activated a similar set of differentially expressed transcripts in kidney and intestine although with some differences in the intensity and time-course response. This set included antiviral-related transcripts (including the mx and interferon-related factors irf1, irf2, irf3, irf7, irf8, irf9, irf10), cytokines (il1b, il6, il8, il12 and tnfa) and their receptors (il1r, il8r, il10r, il15ra, il17r), chemokines (CXC-type, CC-type and IL-8), prostaglandins (cox-2), g-type lysozymes, hepcidin, complement fractions (c2, c4-1 and c4-2) and the antigen differentiation factors cd4, cd8a, and cd8b. The expression profile observed indicated that the host triggered a systemic defensive response including inflammation able to cope with the viral challenge. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  13. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    2009-08-01

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  14. Ganciclovir uptake in human mammary carcinoma cells expressing herpes simplex virus thymidine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Haberkorn, Uwe; Khazaie, Khashayarsha; Morr, Iris; Altmann, Annette; Mueller, Markus; Kaick, Gerhard van

    1998-05-01

    Assessment of suicide enzyme activity would have considerable impact on the planning and the individualization of suicide gene therapy of malignant tumors. This may be done by determining the pharmacokinetics of specific substrates. We generated ganciclovir (GCV)-sensitive human mammary carcinoma cell lines after transfection with a retroviral vector bearing the herpes simplex virus thymidine kinase (HSV-tk) gene. Thereafter, uptake measurements and HPLC analyses were performed up to 48 h in an HSV-tk-expressing cell line and in a wild-type cell line using tritiated GCV. HSV-tk-expressing cells showed higher GCV uptake and phosphorylation than control cells, whereas in wild-type MCF7 cells no phosphorylated GCV was detected. In bystander experiments the total GCV uptake was related to the amount of HSV-tk-expressing cells. Furthermore, the uptake of GCV correlated closely with the growth inhibition (r=0.92). Therefore, the accumulation of specific substrates may serve as an indicator of the HSV-tk activity and of therapy outcome. Inhibition and competition experiments demonstrated slow transport of GCV by the nucleoside carriers. The slow uptake and low affinity to HSV-tk indicate that GCV is not an ideal substrate for the nucleoside transport systems or for HSV-tk. This may be the limiting factor for therapy success, necessitating the search for better substrates of HSV-tk.

  15. Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi

    Directory of Open Access Journals (Sweden)

    Rohde Jörg

    2012-07-01

    Full Text Available Abstract Background The Orf virus (ORFV, a zoonotic Parapoxvirus, causes pustular skin lesions in small ruminants (goat and sheep. Intriguingly, ORFV can repeatedly infect its host, despite the induction of a specific immunity. These immune modulating and immune evading properties are still unexplained. Results Here, we describe that ORFV infection of permissive cells impairs the intracellular transport of MHC class I molecules (MHC I as a result of structural disruption and fragmentation of the Golgi apparatus. Depending on the duration of infection, we observed a pronounced co-localization of MHC I and COP-I vesicular structures as well as a reduction of MHC I surface expression of up to 50%. These subversion processes are associated with early ORFV gene expression and are accompanied by disturbed carbohydrate trimming of post-ER MHC I. The MHC I population remaining on the cell surface shows an extended half-life, an effect that might be partially controlled also by late ORFV genes. Conclusions The presented data demonstrate that ORFV down-regulates MHC I surface expression in infected cells by targeting the late vesicular export machinery and the structure and function of the Golgi apparatus, which might aid to escape cellular immune recognition.

  16. Dynamic Epstein-Barr Virus Gene Expression on the Path to B-Cell Transformation

    Science.gov (United States)

    Price, Alexander M.; Luftig, Micah A.

    2016-01-01

    Epstein-Barr Virus is an oncogenic human herpesvirus in the γ-herpesvirinae sub-family that contains a 170–180 kb double stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B cell compartment of the peripheral blood. EBV can be reactivated from its latent state leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome as well as structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady state viral gene expression within EBV immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection EBV only expressed the well-characterized latency associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation as well as delayed responses in the known latency genes. This review summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, inhibition of apoptosis, and control of innate and adaptive immune responses. PMID:24373315

  17. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  18. Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein.

    Science.gov (United States)

    Baghbani-arani, F; Roohvand, F; Aghasadeghi, M R; Eidi, A; Amini, S; Motevalli, F; Sadat, S M; Memarnejadian, A; Khalili, G

    2012-01-01

    Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.

  19. Infection of primary CD4+ and CD8+ T lymphocytes by Epstein-Barr virus enhances human immunodeficiency virus expression.

    OpenAIRE

    Guan, M; Zhang, R D; Wu, B; Henderson, E E

    1996-01-01

    CD4+ and CD8+ T lymphocytes purified from normal adult donors by flow cytometry could be infected with Epstein-Barr virus (EBV) as measured by the accumulation of components of the EBV replicative cycle, viral DNA and viral transcripts encoding EBER1 and BRLF1. EBV infection resulted in enhanced replication of human immunodeficiency virus type 1 (HIV-1) IIIB in CD4+ lymphocytes as measured by accumulation of reverse transcriptase and formation of syncytia. Furthermore, a small percentage of C...

  20. Permanent cell lines that show temperature-dependent expression of adenovirus virus-associated RNA.

    Science.gov (United States)

    Martínez, C; de la Luna, S; Peláez, F; Lopez-Turiso, A; Valcárcel, J; de Haro, C; Ortín, J

    1989-01-01

    Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection. Images PMID:2585610

  1. Unique Epstein-Barr virus (EBV) latent gene expression, EBNA promoter usage and EBNA promoter methylation status in chronic active EBV infection.

    OpenAIRE

    Yoshioka, Mikio; Kikuta, Hideaki; Ishiguro, Nobuhisa; Ma, Xiaoming; Kobayashi, Kunihiko

    2003-01-01

    Chronic active Epstein–Barr virus infection (CAEBV) has been considered to be a non-neoplastic T-cell lymphoproliferative disease associated with Epstein–Barr virus (EBV) infection. In EBV-associated diseases, the cell phenotype-dependent differences in EBV latent gene expression may reflect the strategy of the virus in relation to latent infection. We previously reported that EBV latent gene expression was restricted; EBV nuclear antigen 1 (EBNA1) transcripts were consistently detected in al...

  2. Ebola virus infection inversely correlates with the overall expression levels of promyelocytic leukaemia (PML protein in cultured cells

    Directory of Open Access Journals (Sweden)

    Szekely Laszlo

    2003-04-01

    Full Text Available Abstract Background Ebola virus causes severe, often fatal hemorrhagic fever in humans. The mechanism of escape from cellular anti-viral mechanisms is not yet fully understood. The promyelocytic leukaemia (PML associated nuclear body is part of the interferon inducible cellular defense system. Several RNA viruses have been found to interfere with the anti-viral function of the PML body. The possible interaction between Ebola virus and the PML bodies has not yet been explored. Results We found that two cell lines, Vero E6 and MCF7, support virus production at high and low levels respectively. The expression of viral proteins was visualized and quantified using high resolution immunofluorescence microscopy. Ebola encoded NP and VP35 accumulated in cytoplasmic inclusion bodies whereas VP40 was mainly membrane associated but it was also present diffusely in the cytoplasm as well as in the euchromatic areas of the nucleus. The anti-VP40 antibody also allowed the detection of extracellular virions. Interferon-alpha treatment decreased the production of all three viral proteins and delayed the development of cytopathic effects in both cell lines. Virus infection and interferon-alpha treatment induced high levels of PML protein expression in MCF7 but much less in Vero E6 cells. No disruption of PML bodies, a common phenomenon induced by a variety of different viruses, was observed. Conclusion We have established a simple fixation and immunofluorescence staining procedure that allows specific co-detection and precise sub-cellular localization of the PML nuclear bodies and the Ebola virus encoded proteins NP, VP35 and VP40 in formaldehyde treated cells. Interferon-alpha treatment delays virus production in vitro. Intact PML bodies may play an anti-viral role in Ebola infected cells.

  3. Varicella-Zoster Virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    Directory of Open Access Journals (Sweden)

    John Earl Carpenter

    2014-07-01

    Full Text Available Varicella-zoster virus (VZV is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR: XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8 fold roughly half of the array elements while downregulating only three (one ERAD and two FOLD components. VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64 fold as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.

  4. Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins.

    Science.gov (United States)

    Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S

    2018-01-29

    Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c + /CD8 + dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Differential gene expression analysis of in vitro duck hepatitis B virus infected primary duck hepatocyte cultures

    Directory of Open Access Journals (Sweden)

    Issac Aneesh

    2011-07-01

    Full Text Available Abstract Background The human hepatitis B virus (HBV, a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC. The duck hepatitis B virus (DHBV infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference. Findings A subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD, Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study. Conclusions The present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.

  6. Expression and characterization of duck enteritis virus gI gene

    Directory of Open Access Journals (Sweden)

    Zhu Dekang

    2011-05-01

    Full Text Available Abstract Background At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. Results The special 1221-bp fragment containing complete open reading frame(ORF of duck enteritis virus(DEV gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+, resulting in pET-32a(+-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+-gI was transformed into E.coli BL21(DE3 competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG. SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs, the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. Conclusions The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3 and maintained its antigenicity very well. The basic information of the transcription and intracellular

  7. Lymphocytic choriomeningitis virus infection is associated with long-standing perturbation of LFA-1 expression on CD8+ T cells

    DEFF Research Database (Denmark)

    Andersson, E C; Christensen, Jan Pravsgaard; Scheynius, A

    1995-01-01

    belonged to the CD8+LFA-1hi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive......Flow cytometric analysis of splenocytes from mice infected with lymphocytic choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8+, but not on CD4+ T cells. Appearance of CD8+ T cells with a changed expression of adhesion molecules reflected polyclonal...... activation and expansion which was demonstrated not to depend on CD4+ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1hiMEL-14lo), but since about 80% of splenic CD8+ T cells have a changed phenotype, extensive bystander activation must...

  8. Buggy Creek virus (Togaviridae: Alphavirus) upregulates expression of pattern recognition receptors and interferons in House Sparrows (Passer domesticus).

    Science.gov (United States)

    Fassbinder-Orth, Carol A; Barak, Virginia A; Rainwater, Ellecia L; Altrichter, Ashley M

    2014-06-01

    Birds serve as reservoirs for at least 10 arthropod-borne viruses, yet specific immune responses of birds to arboviral infections are relatively unknown. Here, adult House Sparrows were inoculated with an arboviral alphavirus, Buggy Creek virus (BCRV), or saline, and euthanized between 1 and 3 days postinoculation. Virological dynamics and gene expression dynamics were investigated. Birds did not develop viremia postinoculation, but cytopathic virus was found in the skeletal muscle and spleen of birds 1 and 3 days postinoculation (DPI). Viral RNA was detected in the blood of BCRV-infected birds 1 and 2 DPI, in oral swabs 1-3 DPI, and in brain, heart, skeletal muscle, and spleen 1-3 DPI. Multiple genes were significantly upregulated following BCRV infection, including pattern recognition receptors (TLR7, TLR15, RIG-1), type I interferon (IFN-α), and type II interferon (IFN-γ). This is the first study to report avian immunological gene expression profiles following an arboviral infection.

  9. Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus.

    Science.gov (United States)

    Aldabe, R; Feduchi, E; Novoa, I; Carrasco, L

    1995-10-24

    Poliovirus protease 2A cleaves p220, a component of initiation factor eIF-4F. Polyclonal antibodies that recognize p220 and the cleaved products from different species have been raised. Transfection of several cell lines with poliovirus 2Apro cloned in different plasmids leads to efficient cleavage of p220 upon infection with VT7, a recombinant vaccinia virus that expresses the T7 RNA polymerase. Under these conditions vaccinia virus protein synthesis is severely inhibited, while expression of poliovirus protein 2C from a similar plasmid has no effect. These results show by the first time the effects of p220 cleavage on vaccinia virus translation in the infected cells.

  10. Cell Culture Evaluation of the Semliki Forest Virus Expression System As a Novel Approach for Antigen Delivery and Expression in Fish.

    Science.gov (United States)

    Phenix; McKenna; Fitzpatrick; Vaughan; Atkins; Liljestrom; Todd

    2000-01-01

    Heterologous gene expression by Semliki Forest virus (SFV) expression vectors was investigated in fish cell culture. Experiments performed using an infectious strain of SFV, replication-defective SFV particles, and recombinant SFV RNA constructs encoding the Escherchia coli LacZ or firefly luciferase reporter genes indicated that levels of SFV-mediated expression in fish cells were dependent on cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9 at 25 degrees C and 20 degrees C. As the temperature was lowered to 15 degrees C or below, levels of reporter gene expression were reduced up to 1000-fold, indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems to function in fish cells was further investigated by analyzing the expression of the protective VP2 antigen of infectious pancreatic necrosis virus (IPNV) from the various constructs, including a novel DNA-based SFV plasmid. The VP2 protein produced in CHSE-214 and F95/9 cells transfected or infected with the recombinant SFV-IPNV VP2 constructs appeared to be synthesized in an antigenically correct form, as evidenced by the ability to react with several conformation-dependent IPNV-specific monoclonal antibodies. Whether the temperature-restricted replication and expression displayed by SFV-based constructs in fish cell culture also occurs in vivo remains to be determined.

  11. Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae

    OpenAIRE

    Kumar, Manoj; Saravanan, P.; S.K.Jalali

    2015-01-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although ...

  12. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Deshui Liu

    Full Text Available Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41 were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X. Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N

  13. Infectious bursal disease virus recovery from Vero cells transfected with RNA transcripts is enhanced by expression of the structural proteins in trans.

    Science.gov (United States)

    Peters, M A; Lin, T L; Wu, C C

    2005-11-01

    Positive sense RNA transcripts of infectious bursal disease (IBD) virus genome segments A and B have previously been shown to be infectious. In this study we demonstrate that recovery of IBD virus from the transfection of Vero cells with positive sense RNA transcripts of genome segments A and B was enhanced by expression of the viral structural proteins VP2 with VP3 or by expression of viral polyprotein VP243 from DNA plasmids in trans. Expression of individual viral proteins VP2, VP3, or VP4 alone from DNA plasmids did not enhance IBD virus recovery. Earliest virus recovery from transfection of positive sense RNA transcripts of genomic segments A and B was at 36 h and mean titers were 10(1.8) pfu/ml. IBD virus was recovered 6 hours after transfection in cells concurrently expressing either VP2 with VP3 or VP243 and mean titers were 10(8.5) pfu/ml or 10(9.2) pfu/ml, respectively. Likewise, expression of the viral polyprotein from DNA plasmid increased the permissiveness of Vero cells for infection with non-culture adapted IBD virus. The titer of recovered non-culture adapted virus from 10(3.3) pfu/ml to 10(10.3) pfu/ml with expression of the viral polyprotein. This report is the first to describe a reverse genetics model for IBD virus with high efficiency of virus recovery for non-culture adapted strains.

  14. Interleukin 37 expression in mice alters sleep responses to inflammatory agents and influenza virus infection

    Directory of Open Access Journals (Sweden)

    Christopher J. Davis

    2017-06-01

    Full Text Available Multiple interactions between the immune system and sleep are known, including the effects of microbial challenge on sleep or the effects of sleep loss on facets of the immune response. Cytokines regulate, in part, sleep and immune responses. Here we examine the role of an anti-inflammatory cytokine, interleukin-37 (IL-37 on sleep in a mouse strain that expresses human IL-37b (IL37tg mice. Constitutive expression of the IL-37 gene in the brains of these mice under resting conditions is low; however, upon an inflammatory stimulus, expression increases dramatically. We measured sleep in three conditions; (a under baseline conditions and after 6 h of sleep loss, (b after bolus intraperitoneal administration of lipopolysaccharide (LPS or IL-1β and (c after intranasal influenza virus challenge. Under baseline conditions, the IL37tg mice had 7% more spontaneous non-rapid eye movement sleep (NREMS during the light period than wild-type (WT mice. After sleep deprivation both WT mice and IL37tg mice slept an extra 21% and 12%, respectively, during the first 6 h of recovery. NREMS responses after sleep deprivation did not significantly differ between WT mice and IL37tg mice. However, in response to either IL-1β or LPS, the increases in time spent in NREMS were about four-fold greater in the WT mice than in the IL37tg mice. In contrast, in response to a low dose of mouse-adapted H1N1 influenza virus, sleep responses developed slowly over the 6 day recording period. By day 6, NREMS increased by 10% and REMS increased by 18% in the IL37tg mice compared to the WT mice. Further, by day 4 IL37tg mice lost less weight, remained more active, and retained their body temperatures closer to baseline values than WT mice. We conclude that conditions that promote IL-37 expression attenuate morbidity to severe inflammatory challenge.

  15. [Targeted inhibition of Rabies virus gene expression by a chimeric multidomain protein mediated shRNA delivery].

    Science.gov (United States)

    Yang, Ruimei; Wang, Hualei; Shan, Hu; Yang, Songtao; Xia, Xianzhu

    2016-01-04

    In this study, a new chimeric protein SEG expressed in previous work was applied to evaluate its translocating efficiency of shRNA to rabies virus infected cells in mice, meanwhile, the capability of anti-rabies virus was investigated. Rabies virus strain CVS-24 was inoculated into the hind leg to establish a mouse model of rabies in a dose of 50 LD₅₀; 12 h thereafter the mice were injected intravenously with shRNA-producing plasmid mixed with SEG. To test shRNA delivery, single-cell suspensions from brain, spleen and liver were examined by flow cytometry. Rabies virus in brain tissue of mice was detected by qRT-PCR, RT-PCR, western blot and directed immunofluorescence assay. Mice were monitored for survival and serum samples were tested for IFN-α levels. No green fluorescent protein (GFP) was seen in the spleen or liver, suggesting that SEG allows specific targeting of RV-infected cells. RT-PCR and western blot showed that mice treated with SEG-shRNA had lower rabies virus RNA and protein levels than the controls. Real-time PCR showed that rabies virus was reduced 4.88 fold compared to the mock cells. Survival of RV-infected mouse showed a significant protection from rabies virus infection by SEG-shRNA treatment. The survival was up to 50% whereas the control group all died. IFN was not induced in SEG-shRNA treated animals. shRNA-producing plasmid was specifically delivered into rabies virus infected cells using the SEG protein, and effectively inhibited rabies virus geneexpression and replication in vivo. SEG-shRNA can be used for adjuvant treatment for rabies.

  16. Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

    OpenAIRE

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny; Weiss, Daniel J.

    2012-01-01

    Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Reco...

  17. Epstein-Barr virus transcription activator R upregulates BARF1 expression by direct binding to its promoter, independent of methylation

    NARCIS (Netherlands)

    Hoebe, E. K.; Wille, C.; Hopmans, E. S.; Robinson, A. R.; Middeldorp, J. M.; Kenney, S. C.; Greijer, A. E.

    2012-01-01

    Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional

  18. Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4(+)-T-cell lines

    NARCIS (Netherlands)

    van 't Wout, Angélique B.; Lehrman, Ginger K.; Mikheeva, Svetlana A.; O'Keeffe, Gemma C.; Katze, Michael G.; Bumgarner, Roger E.; Geiss, Gary K.; Mullins, James I.

    2003-01-01

    The expression levels of approximately 4,600 cellular RNA transcripts were assessed in CD4(+)-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1(BRU)) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1(BRU)

  19. Yeast expressing hepatitis B virus surface antigen determinants on its surface: Implications for a possible oral vaccine

    NARCIS (Netherlands)

    Schreuder, M.P.; Deen, C.; Boersma, W.J.A.; Pouwels, P.H.; Klis, F.M.

    1996-01-01

    The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of 'Bakers Yeast', Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast α-agglutinin carboxyterminal

  20. Molecular cloning and expression of full-length DNA copies of the genomic RNAs of cowpea mosaic virus

    NARCIS (Netherlands)

    Vos, P.

    1987-01-01

    The experiments described in this thesis were designed to unravel various aspects of the mechanism of gene expression of cowpea mosaic virus (CPMV). For this purpose full-length DNA copies of both genomic RNAs of CPMV were constructed. Using powerful invitro

  1. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

    Directory of Open Access Journals (Sweden)

    Lolita George Mathew

    2014-01-01

    Full Text Available Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs. In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

  2. Infection of apple by apple stem grooving virus leads to extensive alterations in gene expression patterns but no disease symptoms.

    Directory of Open Access Journals (Sweden)

    Shanyi Chen

    Full Text Available To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV, which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question of whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoot cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Thus, global host gene expression changes do not necessarily lead to virus disease symptoms. Our data suggest that the general approaches to correlate host gene expression changes under viral infection conditions to specific disease symptom, based on the interpretation of transcription profiling data and altered individual gene functions, may have limitations depending on particular experimental systems.

  3. Infection of apple by apple stem grooving virus leads to extensive alterations in gene expression patterns but no disease symptoms.

    Science.gov (United States)

    Chen, Shanyi; Ye, Ting; Hao, Lu; Chen, Hui; Wang, Shaojie; Fan, Zaifeng; Guo, Liyun; Zhou, Tao

    2014-01-01

    To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question of whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoot cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Thus, global host gene expression changes do not necessarily lead to virus disease symptoms. Our data suggest that the general approaches to correlate host gene expression changes under viral infection conditions to specific disease symptom, based on the interpretation of transcription profiling data and altered individual gene functions, may have limitations depending on particular experimental systems.

  4. Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

    Directory of Open Access Journals (Sweden)

    Saki Kondo

    Full Text Available Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs, are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

  5. Recombinant vaccinia DIs expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate.

    Science.gov (United States)

    Okamura, Tomotaka; Someya, Kenji; Matsuo, Kazuhiro; Hasegawa, Atsuhiko; Yamamoto, Naoki; Honda, Mitsuo

    2006-01-01

    A highly attenuated vaccinia virus substrain of Dairen-I (DIs) shows promise as a candidate vector for eliciting positive immunity against immune deficiency virus. DIs was randomly obtained by serial 1-day egg passages of a chorioarantoic membrane-adapted Dairen strain (DIE), resulting in substantial genomic deletion, including various genes regulating the virus-host-range. To investigate the impact of that deletion and of the subsequent insertion of a foreign gene into that region of DIs on the ability of the DIs recombinant to induce antigen-specific immunity, we generated a recombinant vaccinia DIs expressing fulllength gag and pol genes of simian immunodeficiency virus (SIV) (rDIsSIV gag/pol) and studied the biological and immunological characteristics of the recombinant natural mutant. The rDIsSIV gag/pol developed a tiny plaque on the chick embryo fibroblast (CEF). Viral particles of rDIsSIV gag/pol as well as SIV Gag-like particles were electromicroscopically detected in the cytoplasm. Interestingly, the recombinant DIs strain grows well in CEF cells but not in mammalian cells. While rDIsSIV gag/pol produces SIV proteins in mammalian HeLa and CV-1 cells, recombinant modified vaccinia Ankara strain (MVA) expressing SIV gag and pol genes (MVA/SIV239 gag/pol) clearly replicates in HeLa and CV-1 cell lines under synchronized growth conditions and produces the SIV protein in all cell lines. Moreover, intradermal administration of rDIsSIV gag/pol or of MVA/SIV239 gag/pol elicited similar levels of IFN-gamma spot-forming cells specific for SIV Gag. If the non-productive infection characteristically induced by recombinant DIs is sufficient to trigger immune induction, as we believe it is, then a human immunodeficiency virus vaccine employing the DIs recombinant would have the twin advantages of being both effective and safe.

  6. Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

    Science.gov (United States)

    Krüger, Nadine; Sauder, Christian; Hoffmann, Markus; Örvell, Claes; Drexler, Jan Felix; Rubin, Steven; Herrler, Georg

    2016-11-01

    A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

  7. Dendritic cell subtypes from lymph nodes and blood show contrasted gene expression programs upon Bluetongue virus infection.

    Science.gov (United States)

    Ruscanu, Suzana; Jouneau, Luc; Urien, Céline; Bourge, Mickael; Lecardonnel, Jérôme; Moroldo, Marco; Loup, Benoit; Dalod, Marc; Elhmouzi-Younes, Jamila; Bevilacqua, Claudia; Hope, Jayne; Vitour, Damien; Zientara, Stéphan; Meyer, Gilles; Schwartz-Cornil, Isabelle

    2013-08-01

    Human and animal hemorrhagic viruses initially target dendritic cells (DCs). It has been proposed, but not documented, that both plasmacytoid DCs (pDCs) and conventional DCs (cDCs) may participate in the cytokine storm encountered in these infections. In order to evaluate the contribution of DCs in hemorrhagic virus pathogenesis, we performed a genome-wide expression analysis during infection by Bluetongue virus (BTV), a double-stranded RNA virus that induces hemorrhagic fever in sheep and initially infects cDCs. Both pDCs and cDCs accumulated in regional lymph nodes and spleen during BTV infection. The gene response profiles were performed at the onset of the disease and markedly differed with the DC subtypes and their lymphoid organ location. An integrative knowledge-based analysis revealed that blood pDCs displayed a gene signature related to activation of systemic inflammation and permeability of vasculature. In contrast, the gene profile of pDCs and cDCs in lymph nodes was oriented to inhibition of inflammation, whereas spleen cDCs did not show a clear functional orientation. These analyses indicate that tissue location and DC subtype affect the functional gene expression program induced by BTV and suggest the involvement of blood pDCs in the inflammation and plasma leakage/hemorrhage during BTV infection in the real natural host of the virus. These findings open the avenue to target DCs for therapeutic interventions in viral hemorrhagic diseases.

  8. A Semliki forest virus vector engineered to express IFNα induces efficient elimination of established tumors.

    Science.gov (United States)

    Quetglas, J I; Fioravanti, J; Ardaiz, N; Medina-Echeverz, J; Baraibar, I; Prieto, J; Smerdou, C; Berraondo, P

    2012-03-01

    Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNα). IFNα displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNα, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNα both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNα by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.

  9. Inhibition of hepatitis B virus gene expression and replication by ribonuclease P.

    Science.gov (United States)

    Xia, Chuan; Chen, Yuan-Chuan; Gong, Hao; Zeng, Wenbo; Vu, Gia-Phong; Trang, Phong; Lu, Sangwei; Wu, Jianguo; Liu, Fenyong

    2013-05-01

    Nucleic acid-based gene interfering approaches, such as those mediated by RNA interference and RNase P-associated external guide sequence (EGS), have emerged as promising antiviral strategies. The RNase P-based technology is unique, because a custom-designed EGS can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, a functional EGS was constructed to target hepatitis B virus (HBV) essential transcripts. Furthermore, an attenuated Salmonella strain was constructed and used for delivery of anti-HBV EGS in cells and in mice. Substantial reduction in the levels of HBV gene expression and viral DNA was detected in cells treated with the Salmonella vector carrying the functional EGS construct. Furthermore, oral inoculation of Salmonella carrying the EGS construct led to an inhibition of ~95% in the levels of HBV gene expression and a reduction of ~200,000-fold in viral DNA level in the livers and sera of the treated mice transfected with a HBV plasmid. Our results suggest that EGSs are effective in inhibiting HBV replication in cultured cells and mammalian livers, and demonstrate the use of Salmonella-mediated delivery of EGS as a promising therapeutic approach for human diseases including HBV infection.

  10. Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion.

    Science.gov (United States)

    Haberman, Rebecca P; Samulski, R Jude; McCown, Thomas J

    2003-08-01

    Seizure disorders present an attractive gene therapy target, particularly because viral vectors such as adeno-associated virus (AAV) and lentivirus can stably transduce neurons. When we targeted the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor with an AAV-delivered antisense oligonucleotide, however, the promoter determined whether focal seizure sensitivity was significantly attenuated or facilitated. One potential means to circumvent this liability would be to express an inhibitory neuroactive peptide and constitutively secrete the peptide from the transduced cell. The neuropeptide galanin can modulate seizure activity in vivo, and the laminar protein fibronectin is usually secreted through a constitutive pathway. Initially, inclusion of the fibronectin secretory signal sequence (FIB) in an AAV vector caused significant gene product secretion in vitro. More importantly, the combination of this secretory signal with the coding sequence for the active galanin peptide significantly attenuated in vivo focal seizure sensitivity, even with different promoters, and prevented kainic acid-induced hilar cell death. Thus, neuroactive peptide expression and local secretion provides a new gene therapy platform for the treatment of neurological disorders.

  11. High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

    Science.gov (United States)

    Gonzalez, Sandra Milena; Taborda, Natalia Andrea; Feria, Manuel Gerónimo; Arcia, David; Aguilar-Jiménez, Wbeimar; Zapata, Wildeman; Rugeles, María Teresa

    2015-01-01

    Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i) one of 58 HIV-exposed seronegative individuals (HESNs) who were compared with 59 healthy controls (HCs), and ii) another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT) samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR. HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs), oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT. These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

  12. High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

    Directory of Open Access Journals (Sweden)

    Sandra Milena Gonzalez

    Full Text Available Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i one of 58 HIV-exposed seronegative individuals (HESNs who were compared with 59 healthy controls (HCs, and ii another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR.HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs, oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT.These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

  13. Overcoming maternal antibody interference by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of swine influenza virus.

    Science.gov (United States)

    Wesley, Ronald D; Lager, Kelly M

    2006-11-26

    Sows and gilts lack immunity to human adenovirus 5 (Ad-5) vectored vaccines so immunogens of swine pathogens can be expressed with these vaccines in order to immunize suckling piglets that have interfering, maternally derived antibodies. In this study 7-day-old piglets, that had suckled H3N2 infected gilts, were sham-inoculated with a non-expressing Ad-5 vector or given a primary vaccination with replication-defective Ad-5 viruses expressed the H3 hemagglutinin and the nucleoprotein of swine influenza virus (SIV) subtype H3N2. The hemagglutination inhibition (HI) titer of the sham-inoculated group (n = 12) showed continued antibody decay whereas piglets vaccinated with Ad-5 SIV (n = 23) developed an active immune response by the second week post-vaccination. At 4 weeks-of-age when the HI titer of the sham-inoculated group had decayed to 45, the sham-inoculated group and half of the Ad-5 SIV vaccinated pigs were boosted with a commercial inactivated SIV vaccine. The boosted pigs that had been primed in the presence of maternal interfering antibodies had a strong anamnestic response while sham-inoculated pigs did not respond to the commercial vaccine. Two weeks after the booster vaccination the pigs were challenged with a non-homologous H3N2 virulent SIV. The efficacy of the vaccination protocol was demonstrated by abrogation of clinical signs, by clearance of challenge virus from pulmonary lavage fluids, by markedly reduced virus shedding in nasal secretions, and by the absence of moderate or severe SIV-induced lung lesions. These recombinant Ad-5 SIV vaccines are useful for priming the immune system to override the effects of maternally derived antibodies which interfere with conventional SIV vaccines.

  14. Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection

    Directory of Open Access Journals (Sweden)

    Griffiths Jonathan S

    2008-07-01

    Full Text Available Abstract Background Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV. To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled. Results Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q ≤ 0.05 up- (≥ 2.5 fold and downregulated (≤ -2.5 fold, respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes

  15. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

    Science.gov (United States)

    Purcell, Maureen K.; Kurath, Gael; Garver, Kyle A.; Herwig, Russell P.; Winton, James R.

    2004-01-01

    Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

  16. Recombinant influenza virus expressing a fusion protein neutralizing epitope of respiratory syncytial virus (RSV) confers protection without vaccine-enhanced RSV disease.

    Science.gov (United States)

    Lee, Yu-Na; Hwang, Hye Suk; Kim, Min-Chul; Lee, Young-Tae; Lee, Jong Seok; Moore, Martin L; Kang, Sang-Moo

    2015-03-01

    Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis in both children and the elderly. There is no vaccine available for the prevention of RSV infection. Here, we generated recombinant influenza virus (PR8/RSV.HA-F) expressing an RSV F243-294 neutralizing epitope in the hemagglutinin (HA) as a chimeric protein. Neutralizing antibodies specific for both RSV and influenza virus were induced by a single intranasal immunization of mice with PR8/RSV.HA-F. Mice that were immunized with PR8/RSV.HA-F were protected against RSV infection comparable with live RSV as evidenced by significant reduction of RSV lung viral loads, as well as the absence of lung eosinophilia and RSV-specific cellular immune responses. In contrast, formalin-inactivated RSV-immunized mice showed severe disease and high cellular immune responses in lungs after RSV infection. These findings support a concept that recombinant influenza virus carrying the RSV F243-294 neutralizing epitope can be developed as a promising RSV vaccine candidate which induces protective neutralizing antibodies but avoids lung immunopathology. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus.

    Science.gov (United States)

    Shim, Byoung-Shik; Choi, Jung-Ah; Song, Ho-Hyun; Park, Sung-Moo; Cheon, In Su; Jang, Ji-Eun; Woo, Sun Je; Cho, Chung Hwan; Song, Min-Suk; Kim, Hyemi; Song, Kyung Joo; Lee, Jae Myun; Kim, Suhng Wook; Song, Dae Sub; Choi, Young Ki; Kim, Jae-Ouk; Nguyen, Huan Huu; Kim, Dong Wook; Bahk, Young Yil; Yun, Cheol-Heui; Song, Man Ki

    2013-02-01

    Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.

  18. Identifying protective host gene expression signatures within the spleen during West Nile virus infection in the collaborative cross model

    Directory of Open Access Journals (Sweden)

    Richard Green

    2016-12-01

    Full Text Available Flaviviruses are hematophagous arthropod-viruses that pose global challenges to human health. Like Zika virus, West Nile Virus (WNV is a flavivirus for which no approved vaccine exists [1]. The role host genetics play in early detection and response to WNV still remains largely unexplained. In order to capture the impact of genetic variation on innate immune responses, we studied gene expression following WNV infection using the collaborative cross (CC. The CC is a mouse genetics resource composed of hundreds of independently bred, octo-parental recombinant inbred mouse lines [2]. To accurately capture the host immune gene expression signatures of West Nile infection, we used the nanostring platform to evaluate expression in spleen tissue isolated from CC mice infected with WNV over a time course of 4, 7, and 12 days' post-infection [3]. Nanostring is a non-amplification based digital method to quantitate gene expression that uses color-coded molecular barcodes to detect hundreds of transcripts in a sample. Using this approach, we identified unique gene signatures in spleen tissue at days 4, 7, and 12 following WNV infection, which delineated distinct differences between asymptomatic and symptomatic CC lines. We also identified novel immune genes. Data was deposited into the Gene Expression Omnibus under accession GSE86000.

  19. Identifying protective host gene expression signatures within the spleen during West Nile virus infection in the collaborative cross model.

    Science.gov (United States)

    Green, Richard; Wilkins, Courtney; Thomas, Sunil; Sekine, Aimee; Ireton, Renee C; Ferris, Martin T; Hendrick, Duncan M; Voss, Kathleen; de Villena, Fernando Pardo-Manuel; Baric, Ralph; Heise, Mark; Gale, Michael

    2016-12-01

    Flaviviruses are hematophagous arthropod-viruses that pose global challenges to human health. Like Zika virus, West Nile Virus (WNV) is a flavivirus for which no approved vaccine exists [1]. The role host genetics play in early detection and response to WNV still remains largely unexplained. In order to capture the impact of genetic variation on innate immune responses, we studied gene expression following WNV infection using the collaborative cross (CC). The CC is a mouse genetics resource composed of hundreds of independently bred, octo-parental recombinant inbred mouse lines [2]. To accurately capture the host immune gene expression signatures of West Nile infection, we used the nanostring platform to evaluate expression in spleen tissue isolated from CC mice infected with WNV over a time course of 4, 7, and 12 days' post-infection [3]. Nanostring is a non-amplification based digital method to quantitate gene expression that uses color-coded molecular barcodes to detect hundreds of transcripts in a sample. Using this approach, we identified unique gene signatures in spleen tissue at days 4, 7, and 12 following WNV infection, which delineated distinct differences between asymptomatic and symptomatic CC lines. We also identified novel immune genes. Data was deposited into the Gene Expression Omnibus under accession GSE86000.

  20. Proteomic analysis of differentially expressed proteins in hepatitis B virus-related hepatocellular carcinoma tissues

    Directory of Open Access Journals (Sweden)

    Li Cui

    2009-08-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC, a major cause of cancer death in China, is preceded by chronic hepatitis and liver cirrhosis (LC. Although hepatitis B virus (HBV has been regarded as a clear etiology of human hepatocarcinogenesis, the mechanism is still needs to be further clarified. In this study, we used a proteomic approach to identify the differential expression protein profiles between HCC and the adjacent non-tumorous liver tissues. Methods Eighteen cases of HBV-related HCC including 12 cases of LC-developed HCC and 6 cases of chronic hepatitis B (CHB-developed HCC were analyzed by two-dimensional electrophoresis (2-DE combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, and the results were compared to those of paired adjacent non-tumorous liver tissues. Results A total of 17 differentially expressed proteins with diverse biological functions were identified. Among these, 10 proteins were up-regulated, whereas the other 7 proteins were down-regulated in cancerous tissues. Two proteins, c-Jun N-terminal kinase 2 and ADP/ATP carrier protein were found to be up-regulated only in CHB-developed HCC tissues. Insulin-like growth factor binding protein 2 and Rho-GTPase-activating protein 4 were down-regulated in LC-developed and CHB-developed HCC tissues, respectively. Although 11 out of these 17 proteins have been already described by previous studies, or are already known to be involved in hepatocarcinogenesis, this study revealed 6 new proteins differentially expressed in HBV-related HCC. Conclusion These findings elucidate that there are common features between CHB-developed HCC and LC-developed HCC. The identified proteins are valuable for studying the hepatocarcinogenesis, and may be potential diagnostic markers or therapeutic targets for HBV-related HCC.

  1. Proteomic analysis of differentially expressed proteins in hepatitis B virus-related hepatocellular carcinoma tissues.

    Science.gov (United States)

    Li, Ning; Long, Yunzhu; Fan, Xuegong; Liu, Hongbo; Li, Cui; Chen, Lizhang; Wang, Zhiming

    2009-08-28

    Hepatocellular carcinoma (HCC), a major cause of cancer death in China, is preceded by chronic hepatitis and liver cirrhosis (LC). Although hepatitis B virus (HBV) has been regarded as a clear etiology of human hepatocarcinogenesis, the mechanism is still needs to be further clarified. In this study, we used a proteomic approach to identify the differential expression protein profiles between HCC and the adjacent non-tumorous liver tissues. Eighteen cases of HBV-related HCC including 12 cases of LC-developed HCC and 6 cases of chronic hepatitis B (CHB)-developed HCC were analyzed by two-dimensional electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and the results were compared to those of paired adjacent non-tumorous liver tissues. A total of 17 differentially expressed proteins with diverse biological functions were identified. Among these, 10 proteins were up-regulated, whereas the other 7 proteins were down-regulated in cancerous tissues. Two proteins, c-Jun N-terminal kinase 2 and ADP/ATP carrier protein were found to be up-regulated only in CHB-developed HCC tissues. Insulin-like growth factor binding protein 2 and Rho-GTPase-activating protein 4 were down-regulated in LC-developed and CHB-developed HCC tissues, respectively. Although 11 out of these 17 proteins have been already described by previous studies, or are already known to be involved in hepatocarcinogenesis, this study revealed 6 new proteins differentially expressed in HBV-related HCC. These findings elucidate that there are common features between CHB-developed HCC and LC-developed HCC. The identified proteins are valuable for studying the hepatocarcinogenesis, and may be potential diagnostic markers or therapeutic targets for HBV-related HCC.

  2. Hepatitis C Virus Driven AXL Expression Suppresses the Hepatic Type I Interferon Response.

    Directory of Open Access Journals (Sweden)

    Scott A Read

    Full Text Available Treatment of chronic hepatitis C virus (HCV infection is evolving rapidly with the development of novel direct acting antivirals (DAAs, however viral clearance remains intimately linked to the hepatic innate immune system. Patients demonstrating a high baseline activation of interferon stimulated genes (ISGs, termed interferon refractoriness, are less likely to mount a strong antiviral response and achieve viral clearance when placed on treatment. As a result, suppressor of cytokine signalling (SOCS 3 and other regulators of the IFN response have been identified as key candidates for the IFN refractory phenotype due to their regulatory role on the IFN response. AXL is a receptor tyrosine kinase that has been identified as a key regulator of interferon (IFN signalling in myeloid cells of the immune system, but has not been examined in the context of chronic HCV infection. Here, we show that AXL is up-regulated following HCV infection, both in vitro and in vivo and is likely induced by type I/III IFNs and inflammatory signalling pathways. AXL inhibited type IFNα mediated ISG expression resulting in a decrease in its antiviral efficacy against HCV in vitro. Furthermore, patients possessing the favourable IFNL3 rs12979860 genotype associated with treatment response, showed lower AXL expression in the liver and a stronger induction of AXL in the blood, following their first dose of IFN. Together, these data suggest that elevated AXL expression in the liver may mediate an IFN-refractory phenotype characteristic of patients possessing the unfavourable rs12979860 genotype, which is associated with lower rates of viral clearance.

  3. Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

    NARCIS (Netherlands)

    Feenstra, F.; Gennip, van H.G.P.; Maris-Veldhuis, M.A.; Verheij, E.; Rijn, van P.A.

    2014-01-01

    Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially

  4. Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge.

    Science.gov (United States)

    Bhat, S A; Saravanan, P; Hosamani, M; Basagoudanavar, S H; Sreenivasa, B P; Tamilselvan, R P; Venkataramanan, R

    2013-12-01

    Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

    Science.gov (United States)

    Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Kwon, Suk-Yoon; Moon, Jae Sun

    2016-05-01

    We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. General and family-specific gene expression responses to viral hemorrhagic septicaemia virus infection in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Jørgensen, H. B. H.; Sørensen, P.; Cooper, G. A.

    2011-01-01

    tested it by examining gene expression levels in the head kidney of trout at a genome-wide scale with a 16K cDNA microarray for salmonids. Expression levels were recorded during 16 days following bath challenge. The challenge experiment included a relatively low susceptibility (32% survival following...... challenge) and a relatively high susceptibility (18% survival following challenge) trout family that were both split into a group exposed to virus and a non-exposed control group. In total, 939 genes were differentially expressed between infected and non-infected fish (FDR p = 0.05). Five groups of Gene...... over-represented among the 642 differentially expressed genes in the low-susceptibility trout family but not among the 556 differentially expressed genes in the high-susceptibility trout family. Expression profiles for most immune genes discussed showed increased transcription from day 3 post-challenge...

  7. Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

    Science.gov (United States)

    Dinh, Phat X.; Das, Anshuman; Franco, Rodrigo

    2013-01-01

    The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the family of hnRNPs and was recently shown in a genome-wide small interfering RNA (siRNA) screen to support vesicular stomatitis virus (VSV) growth. To decipher the role of hnRNP K in VSV infection, we conducted studies which suggest that the protein is required for VSV spreading. Virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnRNP K, whereas viral genome transcription and replication were reduced slightly. These results indicate that hnRNP K is likely involved in virus assembly and/or release from infected cells. Further studies showed that hnRNP K suppresses apoptosis of virus-infected cells, resulting in increased cell survival during VSV infection. The increased survival of the infected cells was found to be due to the suppression of proapoptotic proteins such as Bcl-XS and Bik in a cell-type-dependent manner. Additionally, depletion of hnRNP K resulted in not only significantly increased levels of T-cell-restricted intracellular antigen 1 (TIA1) but also switching of the expression of the two isoforms of the protein (TIA1a and TIA1b), both of which inhibited VSV replication. hnRNP K was also found to support expression of several cellular proteins known to be required for VSV infection. Overall, our studies demonstrate hnRNP K to be a multifunctional protein that supports VSV infection via its role(s) in suppressing apoptosis of infected cells, inhibiting the expression of antiviral proteins, and maintaining the expression of proteins required for the virus. PMID:23843646

  8. Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae.

    Science.gov (United States)

    Kumar, Manoj; Saravanan, P; Jalali, S K

    2016-03-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7 days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47 kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.

  9. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless.

    Science.gov (United States)

    Saxena, Pooja; Hsieh, Yi-Cheng; Alvarado, Veria Y; Sainsbury, Frank; Saunders, Keith; Lomonossoff, George P; Scholthof, Herman B

    2011-08-01

    Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd. No claim to original US government works.

  10. Measles vaccine expressing the secreted form of West Nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of West Nile virus infection.

    Science.gov (United States)

    Brandler, Samantha; Marianneau, Philippe; Loth, Philippe; Lacôte, Sandra; Combredet, Chantal; Frenkiel, Marie-Pascale; Desprès, Philippe; Contamin, Hugues; Tangy, Frédéric

    2012-07-15

    West Nile virus (WNV) is a mosquito-borne flavivirus that emerged in North America and caused numerous cases of human encephalitis, thus urging the development of a vaccine. We previously demonstrated the efficacy of a recombinant measles vaccine (MV) expressing the secreted form of the envelope glycoprotein from WNV to prevent WNV encephalitis in mice. In the present study, we investigated the capacity of this vaccine candidate to control WNV infection in a primate model. We first established experimental WNV infection of squirrel monkeys (Saimiri sciureus). A high titer of virus was detected in plasma on day 2 after infection, and viremia persisted for 5 days. A single immunization of recombinant MV-WNV vaccine elicited anti-WNV neutralizing antibodies that strongly reduced WNV viremia at challenge. This study demonstrates for the first time the capacity of a recombinant live attenuated measles vector to protect nonhuman primates from a heterologous infectious challenge.

  11. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...... of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids...

  12. The Effect of West Nile Virus Infection on the Midgut Gene Expression of Culex pipiens quinquefasciatus Say (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Chelsea T. Smartt

    2016-12-01

    Full Text Available The interaction of the mosquito and the invading virus is complex and can result in physiological and gene expression alterations in the insect. The association of West Nile virus (WNV and Culex pipiens quinquefasciatus mosquitoes results in measurable changes in gene expression; 22 gene products were shown previously to have altered expression. Sequence analysis of one product, CQ G1A1, revealed 100% amino acid identity to gram negative bacteria binding proteins (CPQGBP in Cx. p. quinquefasciatus, Aedes aegypti (70% and Anopheles gambiae (63% that function in pathogen recognition. CQ G1A1 also was differentially expressed following WNV infection in two populations of Cx. p. quinquefasciatus colonized from Florida with known differences in vector competence for WNV and showed spatial and temporal gene expression differences in midgut, thorax, and carcass tissues. These data suggest gene expression of CQ G1A1 is influenced by WNV infection and the WNV infection-controlled expression differs between populations and tissues.

  13. Plum pox virus induces differential gene expression in the partially resistant stone fruit tree Prunus armeniaca cv. Goldrich.

    Science.gov (United States)

    Schurdi-Levraud Escalettes, Valérie; Hullot, Clémence; Wawrzy'nczak, Danuta; Mathieu, Elodie; Eyquard, Jean-Philippe; Le Gall, Olivier; Decroocq, Véronique

    2006-06-07

    We investigated the changes in the expression profiles of the partially resistant apricot (Prunus armeniaca L.) cultivar Goldrich following inoculation with Plum pox virus (PPV) using cDNA-amplification fragment length polymorphism (AFLP). Altered expression patterns were detected and twenty-one differentially expressed cDNA had homologies with genes in databases coding for proteins involved in metabolism, signal transduction, defense, stress and intra/intercellular connections. Seven of the modified expressed patterns were further investigated by semi-quantitative RT-PCR or Northern blotting. The expression patterns of five of these genes were confirmed in the partially resistant P. armeniaca cv. 'Goldrich' and assessed in a susceptible genotype. One of these cDNAs, coding for a putative class III chitinase, appeared to be repressed in infected plants of the partially resistant genotype and expressed in the susceptible one which could be related to the partially resistant phenotype. On the contrary, the expression patterns of the genes coding for a transketolase, a kinesin-like and an ankyrin-like protein, were clearly linked to the susceptible interaction. These candidate genes could play a role either in the compatible interaction leading to virus invasion or to the quantitative resistance of apricot to PPV.

  14. Meta-Analysis of Aedes aegypti Expression Datasets: Comparing Virus Infection and Blood-Fed Transcriptomes to Identify Markers of Virus Presence

    Directory of Open Access Journals (Sweden)

    Kiyoshi Ferreira Fukutani

    2018-01-01

    Full Text Available The mosquito Aedes aegypti (L. is vector of several arboviruses including dengue, yellow fever, chikungunya, and more recently zika. Previous transcriptomic studies have been performed to elucidate altered pathways in response to viral infection. However, the intrinsic coupling between alimentation and infection were unappreciated in these studies. Feeding is required for the initial mosquito contact with the virus and these events are highly dependent. Addressing this relationship, we reinterrogated datasets of virus-infected mosquitoes with two different diet schemes (fed and unfed mosquitoes, evaluating the metabolic cross-talk during both processes. We constructed coexpression networks with the differentially expressed genes of these comparison: virus-infected versus blood-fed mosquitoes and virus-infected versus unfed mosquitoes. Our analysis identified one module with 110 genes that correlated with infection status (representing ~0.7% of the A. aegypti genome. Furthermore, we performed a machine-learning approach and summarized the infection status using only four genes (AAEL012128, AAEL014210, AAEL002477, and AAEL005350. While three of the four genes were annotated as hypothetical proteins, AAEL012128 gene is a membrane amino acid transporter correlated with viral envelope binding. This gene alone is able to discriminate all infected samples and thus should have a key role to discriminate viral infection in the A. aegypti mosquito. Moreover, validation using external datasets found this gene as differentially expressed in four transcriptomic experiments. Therefore, these genes may serve as a proxy of viral infection in the mosquito and the others 106 identified genes provides a framework to future studies.

  15. Meta-Analysis ofAedes aegyptiExpression Datasets: Comparing Virus Infection and Blood-Fed Transcriptomes to Identify Markers of Virus Presence.

    Science.gov (United States)

    Fukutani, Kiyoshi Ferreira; Kasprzykowski, José Irahe; Paschoal, Alexandre Rossi; Gomes, Matheus de Souza; Barral, Aldina; de Oliveira, Camila I; Ramos, Pablo Ivan Pereira; de Queiroz, Artur Trancoso Lopo

    2017-01-01

    The mosquito Aedes aegypti (L.) is vector of several arboviruses including dengue, yellow fever, chikungunya, and more recently zika. Previous transcriptomic studies have been performed to elucidate altered pathways in response to viral infection. However, the intrinsic coupling between alimentation and infection were unappreciated in these studies. Feeding is required for the initial mosquito contact with the virus and these events are highly dependent. Addressing this relationship, we reinterrogated datasets of virus-infected mosquitoes with two different diet schemes (fed and unfed mosquitoes), evaluating the metabolic cross-talk during both processes. We constructed coexpression networks with the differentially expressed genes of these comparison: virus-infected versus blood-fed mosquitoes and virus-infected versus unfed mosquitoes. Our analysis identified one module with 110 genes that correlated with infection status (representing ~0.7% of the A. aegypti genome). Furthermore, we performed a machine-learning approach and summarized the infection status using only four genes (AAEL012128, AAEL014210, AAEL002477, and AAEL005350). While three of the four genes were annotated as hypothetical proteins, AAEL012128 gene is a membrane amino acid transporter correlated with viral envelope binding. This gene alone is able to discriminate all infected samples and thus should have a key role to discriminate viral infection in the A. aegypti mosquito. Moreover, validation using external datasets found this gene as differentially expressed in four transcriptomic experiments. Therefore, these genes may serve as a proxy of viral infection in the mosquito and the others 106 identified genes provides a framework to future studies.

  16. Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

    Science.gov (United States)

    A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...

  17. Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana.

    Science.gov (United States)

    Tahmasebi, Amin-Alah; Afsharifar, Alireza

    2017-06-01

    Transient expression of proteins in plants has become a choice to facilitate recombinant protein production with its fast and easy application. On the other hand, host defensive mechanisms have been reported to reduce the efficiency of transient expression in plants. Hence, this study was designed to evaluate the effect of cap analog and Potato virus A helper component proteinase (PVA HC-Pro) on green fluorescent protein (GFP) expression efficiency. N. benthamiana leaves were inoculated with capped or un-capped RNA transcripts of a Turnip crinkle virus (TCV) construct containing a green fluorescent protein reporter gene (TCV-sGFP) in place of its coat protein (CP) ORF. PVA HC-Pro as a viral suppressor of RNA silencing was infiltrated in trans by Agrobacterium tumefaciens, increased the GFP foci diameter to six and even more cells in both capped and un capped treatments. The expression level of GFP in inoculated plants with TCV-sGFP transcript pre-infiltrated with PVA HC-Pro was 12.97-fold higher than the GFP accumulation level in pre-infiltrated leaves with empty plasmid (EP) control. Also, the yield of GFP in inoculated N. benthamiana plants with capped TCV-sGFP transcript pre-infiltrated with EP and PVA HC-Pro was 1.54 and 1.2-fold respectively, greater than the level of GFP expressed without cap analog application at 5 days post inoculation (dpi). In addition, the movement of TCV-sGFP was increased in some cells of inoculated leaves with capped transcripts. Results of this study indicated that PVA HC-Pro and mRNA capping can increase GFP expression and its cell to cell movement in N. benthamiana.

  18. Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

    Science.gov (United States)

    Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

    2010-10-15

    The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

  19. Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli.

    Science.gov (United States)

    Das, Dipankar; Jacobs, Fred; Feldmann, Heinz; Jones, Steven M; Suresh, Mavanur R

    2007-07-01

    Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.

  20. De novo foliar transcriptome of Chenopodium amaranticolor and analysis of its gene expression during virus-induced hypersensitive response.

    Directory of Open Access Journals (Sweden)

    Yongqiang Zhang

    Full Text Available BACKGROUND: The hypersensitive response (HR system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons. BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10(-5. Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. CONCLUSIONS: To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further

  1. Recombinant Canine Distemper Virus Strain Snyder Hill Expressing Green or Red Fluorescent Proteins Causes Meningoencephalitis in the Ferret

    Science.gov (United States)

    Ludlow, M.; Nguyen, D. T.; Silin, D.; Lyubomska, O.; de Vries, R. D.; von Messling, V.; McQuaid, S.; De Swart, R. L.

    2012-01-01

    The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDVSH) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDVSH (rCDVSH) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV5804P and the prototypic wild-type CDVR252 showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDVSH-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis. PMID:22553334

  2. Expression and characterization of UL16 gene from duck enteritis virus

    Directory of Open Access Journals (Sweden)

    Wang Mingshu

    2011-08-01

    Full Text Available Abstract Background Previous studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV. In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in Escherichia coli Rosetta (DE3 induced by isopropy1-β-D-thiogalactopyranoside (IPTG. The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay. Results In our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli Rossetta (DE3 induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay. Conclusions The UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about

  3. Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

    Directory of Open Access Journals (Sweden)

    Reh Juliane

    2011-08-01

    Full Text Available Abstract Background Foamy viruses (FVs unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation. Results Several Prototype FV (PFV Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85PR-RT and p40IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71Gag

  4. Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus

    Directory of Open Access Journals (Sweden)

    Qian Biao

    2007-03-01

    Full Text Available Abstract Background Infectious salmon anaemia (ISA virus (ISAV, an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE protein encoded on segment 6 and fusion (F protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. Results In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1 so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the

  5. Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication.

    Science.gov (United States)

    Wang, Zekun; Shen, Weiran; Cheng, Fang; Deng, Xuefeng; Engelhardt, John F; Yan, Ziying; Qiu, Jianming

    2017-04-15

    -associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies. Copyright © 2017 American Society for Microbiology.

  6. Immunization with a recombinant fowlpox virus expressing a hepatitis C virus core-E1 polyprotein variant, protects mice and African green monkeys (Chlorocebus aethiops sabaeus) against challenge with a surrogate vaccinia virus.

    Science.gov (United States)

    Alvarez-Lajonchere, Liz; Amador-Cañizares, Yalena; Frías, Roberto; Milian, Yoamel; Musacchio, Alexis; Guerra, Ivis; Acosta-Rivero, Nelson; Martínez, Gillian; Castro, Jorge; Puentes, Pedro; Cosme, Karelia; Dueñas-Carrera, Santiago

    2008-10-01

    HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core-E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon-gamma secretion response against HCV core in ex-vivo ELISPOT (enzyme-linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti-HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.

  7. Computational analysis of HIV-1 resistance based on gene expression profiles and the virus-host interaction network.

    Directory of Open Access Journals (Sweden)

    Tao Huang

    Full Text Available A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR and Incremental Feature Selection (IFS methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection.

  8. Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol in mucosal epithelial cells (specifically Caco-2 cell layers and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

  9. Uncoupling uncoating of herpes simplex virus genomes from their nuclear import and gene expression.

    Science.gov (United States)

    Rode, Kathrin; Döhner, Katinka; Binz, Anne; Glass, Mandy; Strive, Tanja; Bauerfeind, Rudolf; Sodeik, Beate

    2011-05-01

    Incoming capsids of herpes simplex virus type 1 (HSV-1) enter the cytosol by fusion of the viral envelopes with host cell membranes and use microtubules and microtubule motors for transport to the nucleus. Upon docking to the nuclear pores, capsids release their genomes into the nucleoplasm. Progeny genomes are replicated in the nucleoplasm and subsequently packaged into newly assembled capsids. The minor capsid protein pUL25 of alphaherpesviruses is required for capsid stabilization after genome packaging and for nuclear targeting of incoming genomes. Here, we show that HSV-1 pUL25 bound to mature capsids within the nucleus and remained capsid associated during assembly and nuclear targeting. Furthermore, we tested potential interactions between parental pUL25 bound to incoming HSV-1 capsids and host factors by competing for such interactions with an experimental excess of cytosolic pUL25. Overexpression of pUL25, GFPUL25, or UL25GFP prior to infection reduced gene expression of HSV-1. Electron microscopy and in situ hybridization studies revealed that an excess of GFPUL25 or UL25GFP prevented efficient nuclear import and/or transcription of parental HSV-1 genomes, but not nuclear targeting of capsids or the uncoating of the incoming genomes at the nuclear pore. Thus, the uncoating of HSV-1 genomes could be uncoupled from their nuclear import and gene expression. Most likely, surplus pUL25 competed with important interactions between the parental capsids, and possibly between authentic capsid-associated pUL25, and cytosolic or nuclear host factors required for functional interaction of the incoming genomes with the nuclear machinery.

  10. Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

    Science.gov (United States)

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2017-01-01

    Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

  11. Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles.

    Science.gov (United States)

    Ho, Thao T; Nguyen, Jasmine T; Liu, Juping; Stanczak, Pawel; Thompson, Aaron A; Yan, Yingzhuo G; Chen, Jasmine; Allerston, Charles K; Dillard, Charles L; Xu, Hao; Shoger, Nicholas J; Cameron, Jill S; Massari, Mark E; Aertgeerts, Kathleen

    2017-05-01

    Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

    Science.gov (United States)

    Chan, Hui-Ting; Chia, Min-Yuan; Pang, Victor Fei; Jeng, Chian-Ren; Do, Yi-Yin; Huang, Pung-Ling

    2013-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  13. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene.

    Science.gov (United States)

    Scorza, R; Ravelonandro, M; Callahan, A M; Cordts, J M; Fuchs, M; Dunez, J; Gonsalves, D

    1994-11-01

    Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

  14. Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants.

    Science.gov (United States)

    Sainsbury, Frank; Liu, Li; Lomonossoff, George P

    2009-01-01

    This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of biocontainment. In both cases, vector constructs modified to contain a foreign gene of interest can be delivered by agroinfiltration to obtain transient expression of the foreign protein. If required, the same constructs can also be used for stable nuclear transformation. Both types of vector have proved effective for the production in plants of a diverse range of proteins including antigens and antibodies.

  15. Virus-based transient expression vectors for woody crops: a new frontier for vector design and use.

    Science.gov (United States)

    Dawson, William O; Folimonova, Svetlana Y

    2013-01-01

    Virus-based expression vectors are commonplace tools for the production of proteins or the induction of RNA silencing in herbaceous plants. This review considers a completely different set of uses for viral vectors in perennial fruit and nut crops, which can be productive for periods of up to 100 years. Viral vectors could be used in the field to modify existing plants. Furthermore, with continually emerging pathogens and pests, viral vectors could express genes to protect the plants or even to treat plants after they become infected. As technologies develop during the life span of these crops, viral vectors can be used for adding new genes as an alternative to pushing up the crop and replanting with transgenic plants. Another value of virus-based vectors is that they add nothing permanently to the environment. This requires that effective and stable viral vectors be developed for specific crops from endemic viruses. Studies using viruses from perennial hosts suggest that these objectives could be accomplished.

  16. Inhalation of nebulized perfluorochemical enhances recombinant adenovirus and adeno-associated virus-mediated gene expression in lung epithelium.

    Science.gov (United States)

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J; Wang, Lili; Gao, Guang Ping; Kolls, Jay K; Bohm, Rudolf; Liggitt, Denny; Weiss, Daniel J

    2012-04-01

    Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (pliquid perflubron, safely enhances lung gene expression.

  17. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

    Directory of Open Access Journals (Sweden)

    Haroldo Cid da Silva Junior

    2016-08-01

    Full Text Available The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV. The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS. The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

  18. Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Wang, Chengbao; Meng, Han; Gao, Yujin; Gao, Hui; Guo, Kangkang; Almazan, Fernando; Sola, Isabel; Enjuanes, Luis; Zhang, Yanming; Abrahamyan, Levon

    2017-08-10

    In order to gain insight into the role of the transcription regulatory sequences (TRSs) in the regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus (PRRSV), the enhanced green fluorescent protein (EGFP) gene, under the control of the different structural gene TRSs, was inserted between the N gene and 3'-UTR of the PRRSV genome and EGFP expression was analyzed for each TRS. TRSs of all the studied structural genes of PRRSV positively modulated EGFP expression at different levels. Among the TRSs analyzed, those of GP2, GP5, M, and N genes highly enhanced EGFP expression without altering replication of PRRSV. These data indicated that structural gene TRSs could be an extremely useful tool for foreign gene expression using PRRSV as a vector.

  19. PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions.

    Science.gov (United States)

    Chakrabarty, Romit; Banerjee, Rituparna; Chung, Sang-Min; Farman, Mark; Citovsky, Vitaly; Hogenhout, Saskia A; Tzfira, Tzvi; Goodin, Michael

    2007-07-01

    Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

  20. Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene.

    Science.gov (United States)

    Souza, Ana Cláudia; Vasques, Raquel Medeiros; Inoue-Nagata, Alice Kazuko; Lacorte, Cristiano; Maldaner, Franciele Roberta; Noronha, Eliane Ferreira; Nagata, Tatsuya

    2013-10-01

    Binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. However, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (PTGS) in the host plants. The aim of this study was to optimize a procedure for expression of norovirus virus-like particles (VLPs) in plants using a binary vector with co-expression of a PTGS suppressor to increase the yield of the target protein. The effects of four plant viral PTGS suppressors on protein expression were evaluated using green fluorescent protein (GFP) as a reporter. Constructs for both GFP and PTGS suppressor genes were co-infiltrated in Nicotiana benthamiana plants, and the accumulation of GFP was evaluated. The most effective PTGS suppressor was the 126K protein of Pepper mild mottle virus. Therefore, this suppressor was selected as the norovirus capsid gene co-expression partner for subsequent studies. The construct containing the major (vp1) and minor capsid (vp2) genes with a 3'UTR produced a greater amount of protein than the construct with the major capsid gene alone. Thus, the vp1-vp2-3'UTR and 126K PTGS suppressor constructs were co-infiltrated at middle scale and VLPs were purified by sucrose gradient centrifugation. Proteins of the expected size, specific to the norovirus capsid antibody, were observed by Western blot. VLPs were observed by transmission electron microscopy. It was concluded that protein expression in a binary vector co-expressed with the 126K PTGS suppressor protein enabled superior expression and assembly of norovirus VLPs.

  1. Engineering, Expression in Transgenic Plants and Characterisation of E559, a Rabies Virus-Neutralising Monoclonal Antibody

    Science.gov (United States)

    van Dolleweerd, Craig J.; Teh, Audrey Y-H.; Banyard, Ashley C.; Both, Leonard; Lotter-Stark, Hester C. T.; Tsekoa, Tsepo; Phahladira, Baby; Shumba, Wonderful; Chakauya, Ereck; Sabeta, Claude T.; Gruber, Clemens; Fooks, Anthony R.; Chikwamba, Rachel K.; Ma, Julian K-C.

    2014-01-01

    Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model. PMID:24511101

  2. Inhibition of hepatitis B virus surface gene expression by antisense oligodeoxynucleotides in a human hepatoma cell line.

    Science.gov (United States)

    Reinis, M; Reinisová, M; Korec, E; Hlozánek, I

    1993-01-01

    We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.

  3. Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine

    Science.gov (United States)

    Mackow, Natalie; Amaro-Carambot, Emérito; Liang, Bo; Surman, Sonja; Lingemann, Matthias; Yang, Lijuan; Collins, Peter L.

    2015-01-01

    ABSTRACT Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (CΔ170) or a temperature-sensitive missense mutation in the L ORF (LY942A). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-CΔ170-F1, -F2, and -F3 and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-CΔ170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo. Among the CΔ170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The CΔ170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIV. We are developing live attenuated RSV and HPIV vaccines for use in virus-naive infants. Live attenuated RSV strains in particular are difficult to develop due to their poor growth and physical instability, but these obstacles could be

  4. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus.

    Science.gov (United States)

    Luo, Jun; Shi, Hehe; Tan, Yeping; Niu, Xuefeng; Long, Teng; Zhao, Jing; Tian, Qin; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-08-17

    Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. High level of transgene expression in primary chronic lymphocytic leukemia cells using helper-virus-free recombinant Epstein-Barr virus vectors.

    Science.gov (United States)

    Wendtner, Clemens-Martin; Kurzeder, Christian; Theiss, Hans D; Kofler, David M; Baumert, Jens; Delecluse, Henri-Jacques; Janz, Annette; Hammerschmidt, Wolfgang; Hallek, Michael

    2003-02-01

    Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells. EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009). Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.

  6. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

    Science.gov (United States)

    Kochinger, Stefanie; Renevey, Nathalie; Hofmann, Martin A; Zimmer, Gert

    2014-06-13

    Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

  7. Transcription factor regulation and cytokine expression following in vitro infection of primary chicken cell culture with low pathogenic avian influenza virus

    Science.gov (United States)

    Avian influenza virus (AIV) induced proinflammatory cytokine expression is believed to contribute to the disease pathogenesis following infection. However, there is limited information on the avian immune response to infection with low pathogenic avian influenza virus (LPAIV). To gain a better under...

  8. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

    Science.gov (United States)

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  9. Expression of innate immune genes, proteins and microRNAs in lung tissue and leukocytes of pigs infected with influenza virus

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Cirera, Susanna; Vasby, Ditte

    This study aimed at providing a better understanding of the involvement of innate immune factors including microRNA (miRNA) in the local and systemic host response to influenza virus infection. Twenty pigs were challenged by influenza A virus subtype H1N2. Expression of miRNA, mRNA and proteins w...

  10. Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection.

    Science.gov (United States)

    Liang, Bo; Munir, Shirin; Amaro-Carambot, Emerito; Surman, Sonja; Mackow, Natalie; Yang, Lijuan; Buchholz, Ursula J; Collins, Peter L; Schaap-Nutt, Anne

    2014-04-01

    A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109-114, 2012; C.-F. Yang et al., Vaccine 31:2822-2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this

  11. Chimeric Bovine/Human Parainfluenza Virus Type 3 Expressing Respiratory Syncytial Virus (RSV) F Glycoprotein: Effect of Insert Position on Expression, Replication, Immunogenicity, Stability, and Protection against RSV Infection

    Science.gov (United States)

    Munir, Shirin; Amaro-Carambot, Emerito; Surman, Sonja; Mackow, Natalie; Yang, Lijuan; Buchholz, Ursula J.; Collins, Peter L.; Schaap-Nutt, Anne

    2014-01-01

    ABSTRACT A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109–114, 2012; C.-F. Yang et al., Vaccine 31:2822–2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. IMPORTANCE The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young

  12. Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis.

    Science.gov (United States)

    Gulati-Sakhuja, Anju; Sears, John L; Nuñez, Alberto; Liu, Hsing-Yeh

    2009-09-01

    Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3mg refolded protein per 200mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.

  13. Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

    Science.gov (United States)

    Kerr, P J; Perkins, H D; Inglis, B; Stagg, R; McLaughlin, E; Collins, S V; Van Leeuwen, B H

    2004-06-20

    Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

  14. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  15. Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector

    Science.gov (United States)

    Haller, Aurelia A.; Miller, Tessa; Mitiku, Misrach; Coelingh, Kathleen

    2000-01-01

    Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39°C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37°C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses. PMID:11090161

  16. Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression.

    Science.gov (United States)

    Soler, Nuria; Fagoaga, Carmen; López, Carmelo; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2015-05-01

    Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23Δ158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  17. Influenza A virus inhibits type I IFN signaling via NF-kappaB-dependent induction of SOCS-3 expression.

    Directory of Open Access Journals (Sweden)

    Eva-K Pauli

    2008-11-01

    Full Text Available The type I interferon (IFN system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1. Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3 protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1 was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

  18. Establishment of a mouse model stably replicating and expressing hepatitis B virus genotype C prevailed in China

    Directory of Open Access Journals (Sweden)

    Yue YANG

    2016-10-01

    Full Text Available Objective  To establish a mouse model stably replicating and expressing hepatitis B virus genotype C (HBV-C prevailed in China. Methods  The recombinant adeno-associated virus rAAV8-1.3HBV-C (adr serotype was transduced into the human hepatocellular carcinoma Huh7 cells in vitro, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. High expression recombinant virus rAAV8-1.3HBV-C was screened and injected via the tail vein into eight C57BL/6 mice (aged 6-8 weeks as the experimental group; meanwhile the previously reported rAAV8-1.3HBV-D (ayw serotype was injected into seven C57BL/6 mice as the control group. HBV DNA load, HBsAg and HBeAg levels in sera were assayed at weeks 2, 3, 5, 7 and 9 post viral injections. Mice were sacrificed 9 weeks post injection and Hematoxylin-eosin (HE staining and immunohistochemistry were performed to evaluate the pathological changes, and HBsAg and HBcAg expressions in the liver tissue. Results  The supernatant HBsAg and HBeAg were detectable in the HuH7 cells 72h after transduction in vitro. The fluorescence quantitative PCR results of the HBV DNA load in serum at week 2, 3, 5, 7, and 9 post viral injection suggested stable in vivo replication of HBV DNA in mice. The serum expression of HBeAg was stable while the serum expression of HBsAg fluctuated. No obvious inflammatory cell infiltration or abnormal structure of liver tissue was observed, while HBsAg and HBcAg expression in the liver tissue were detected for both groups. Conclusion  By in vivo transduction with the recombinant virus rAAV8-1.3HBV-C, a mouse model that stably expressed and replicated HBV-C has been successfully established. DOI: 10.11855/j.issn.0577-7402.2016.10.01

  19. Efficient replication and expression of murine leukemia virus with major deletions in the enhancer region of U3

    DEFF Research Database (Denmark)

    Pedersen, K.; Lovmand, S.; Bonefeld-Jørgensen, Eva Cecilie

    1992-01-01

    The effect of deletions within the enhancer region in the U3 part of the LTR derived from the murine retrovirus Akv was studied. The deletions were stably transmitted through normal virus replication as shown by sequence analysis of cloned polymerase chain reaction product of the cDNA copy...... of the viral RNA. Genetic tagging of the retrovirus with lacO facilitated the analysis. Among the individual mutated LTRs an over 100-fold difference in a transient expression assay was previously detected. This difference was not revealed in studies of viral replication in cell culture, where the expression...

  20. Transcriptomic analyses of genes differentially expressed by high-risk and low-risk human papilloma virus E6 oncoproteins

    OpenAIRE

    Ganguly, Pooja; Ganguly, Niladri

    2015-01-01

    Human papilloma virus is the causative agent for cervical cancer with 99 % of cervical cancer cases containing HPV. The high risk HPV-16, 18 and 31 are the major causative agents. The low risk HPV-6, 11 have been reported to cause penile, laryngeal, bronchogenic and oesophageal cancer. Since E6 oncoprotein is frequently over expressed in cancers, we did gene expression studies to compare between the E6 genes of high-risk (HPV18) or low-risk (HPV11)stably transfected in epithelial cell line EP...

  1. Oncolytic vesicular stomatitis virus expressing interferon-σ has enhanced therapeutic activity

    Directory of Open Access Journals (Sweden)

    Marie-Claude Bourgeois-Daigneault

    2016-01-01

    Full Text Available Oncolytic viruses are known to stimulate the antitumor immune response by specifically replicating in tumor cells. This is believed to be an important aspect of the durable responses observed in some patients and the field is rapidly moving toward immunotherapy. As a further means to engage the immune system, we engineered a virus, vesicular stomatitis virus (VSV, to encode the proinflammatory cytokine interferon-σ. We used the 4T1 mammary adenocarcinoma as well as other murine tumor models to characterize immune responses in tumor-bearing animals generated by treatment with our viruses. The interferon-σ-encoding virus demonstrated greater activation of dendritic cells and drove a more profound secretion of proinflammatory cytokines compared to the parental virus. From a therapeutic point of view, the interferon-σ virus slowed tumor growth, minimized lung tumors, and prolonged survival in several murine tumor models. The improved efficacy was lost in immunocompromized animals; hence the mechanism appears to be T-cell-mediated. Taken together, these results demonstrate the ability of oncolytic viruses to act as immune stimulators to drive antitumor immunity as well as their potential for targeted gene therapy.

  2. Plum pox virus (PPV) genome expression in genetically engineered RNAi plants

    Science.gov (United States)

    An important approach to controlling sharka disease caused by Plum pox virus (PPV) is the development of PPV resistant plants using small interfering RNAs (siRNA) technology. In order to evaluate siRNA induced gene silencing, we studied, based on knowledge of the PPV genome sequence, virus genome t...

  3. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2014-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  4. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  5. RNAseq expression analysis of resistant and susceptible mice after influenza A virus infection identifies novel genes associated with virus replication and important for host resistance to infection.

    Science.gov (United States)

    Wilk, Esther; Pandey, Ashutosh K; Leist, Sarah Rebecca; Hatesuer, Bastian; Preusse, Matthias; Pommerenke, Claudia; Wang, Junxi; Schughart, Klaus

    2015-09-02

    The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suited to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilized a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual mouse after H1N1 infection. We performed a detailed expression analysis to identify (i) correlations between changes in expression of host and virus genes, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between two mouse strains that strongly differ in resistance to influenza infections. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection. Using RNAseq analysis we identified novel genes important for viral replication or the host defense. This study adds further important knowledge to host-pathogen-interactions and suggests additional candidates that are crucial for host susceptibility or survival during influenza A infections.

  6. Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

    Science.gov (United States)

    Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin

    2017-11-15

    Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a

  7. Quantitative analysis of Epstein-Barr virus (EBV)-related gene expression in patients with chronic active EBV infection.

    Science.gov (United States)

    Iwata, Seiko; Wada, Kaoru; Tobita, Satomi; Gotoh, Kensei; Ito, Yoshinori; Demachi-Okamura, Ayako; Shimizu, Norio; Nishiyama, Yukihiro; Kimura, Hiroshi

    2010-01-01

    Chronic active Epstein-Barr virus (CAEBV) infection is a systemic Epstein-Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.

  8. Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

    Science.gov (United States)

    Ocampo, Clara B.; Caicedo, Paola A.; Jaramillo, Gloria; Ursic Bedoya, Raul; Baron, Olga; Serrato, Idalba M.; Cooper, Dawn M.; Lowenberger, Carl

    2013-01-01

    Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain. PMID:23593426

  9. RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Meleri Jones

    2014-01-01

    Full Text Available Varicella zoster virus (VZV is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

  10. [Construction of adeno-associated virus vector containing ANG-1 gene and its expression in pig mesenchymal stem cells].

    Science.gov (United States)

    ZHU, Cheng-chu; CHEN, Shi-lin; LIU, Yu-qing; TANG, Li-jiang; BAO, Wei-guang

    2009-07-01

    To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1 (ANG-1) gene and to express the ANG-1 in targeting cells. ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1, the virus stocks in high titer were harvested. The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously. Titration of rAAVANG-1 stock was 9 X 10(11)v.g/ml. The expression of ANG-1 gene was detected in transfected cells. Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells, 55% cells expressed GFP. The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.

  11. Inhibition of enveloped viruses infectivity by curcumin.

    Directory of Open Access Journals (Sweden)

    Tzu-Yen Chen

    Full Text Available Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter than for the pseudorabies virus (approximately 180 nm and the vaccinia virus (roughly 335 × 200 × 200 nm. These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses.

  12. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    Science.gov (United States)

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-04

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.

    Science.gov (United States)

    De Baets, Sarah; Verhelst, Judith; Van den Hoecke, Silvie; Smet, Anouk; Schotsaert, Michael; Job, Emma R; Roose, Kenny; Schepens, Bert; Fiers, Walter; Saelens, Xavier

    2015-01-01

    The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.

  14. Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

    Science.gov (United States)

    Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

    2014-01-01

    PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

  15. Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells.

    Science.gov (United States)

    Hensel, Michael T; Peng, Tao; Cheng, Anqi; De Rosa, Stephen C; Wald, Anna; Laing, Kerry J; Jing, Lichen; Dong, Lichun; Magaret, Amalia S; Koelle, David M

    2017-10-01

    Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and

  16. Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications.

    Science.gov (United States)

    Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

    2014-11-01

    Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants

  17. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  18. Study on the expression of human lysozyme in oviduct bioreactor mediated by recombinant avian adeno-associated virus.

    Science.gov (United States)

    Wang, A P; Wang, Y J; Wu, S; Zuo, W Y; Guo, C M; Hong, W M; Zhu, S Y

    2017-07-01

    Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins. © 2017 Poultry Science Association Inc.

  19. Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees.

    Directory of Open Access Journals (Sweden)

    Nadja Steinmann

    Full Text Available The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV, one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee's susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.

  20. Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq.

    Science.gov (United States)

    Rubio, Manuel; Rodríguez-Moreno, Luis; Ballester, Ana Rosa; de Moura, Manuel Castro; Bonghi, Claudio; Candresse, Thierry; Martínez-Gómez, Pedro

    2015-02-01

    Differences in gene expression were studied after Plum pox virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5' genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach-PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  1. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  2. Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

    Science.gov (United States)

    Pearce, Lesley A; Yu, Meng; Waddington, Lynne J; Barr, Jennifer A; Scoble, Judith A; Crameri, Gary S; McKinstry, William J

    2015-12-01

    Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  3. Zika virus alters the microRNA expression profile and elicits an RNAi response in Aedes aegypti mosquitoes.

    Directory of Open Access Journals (Sweden)

    Miguel A Saldaña

    2017-07-01

    Full Text Available Zika virus (ZIKV, a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.

  4. Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame.

    Directory of Open Access Journals (Sweden)

    Marijke van Rikxoort

    Full Text Available Oncolytic influenza A viruses with deleted NS1 gene (delNS1 replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15 coding sequence into the viral NS gene segment (delNS1-IL-15. DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1 infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.

  5. Expression and role of the TGF-β family in glial cells infected with Borna disease virus.

    Science.gov (United States)

    Nishino, Yoshii; Murakami, Masaru; Funaba, Masayuki

    2016-02-01

    A previous study revealed that the expression of the Borna disease virus (BDV)-encoding phosphoprotein in glial cells was sufficient to induce neurobehavioral abnormalities resembling Borna disease. To evaluate the involvement of the TGF-β family in BDV-induced changes in cell responses by C6 glial cells, we examined the expression levels of the TGF-β family and effects of inhibiting the TGF-β family pathway in BDV-infected C6 (C6BV) cells. The expression of activin βA and BMP7 was markedly increased in BDV-infected cells. Expression of Smad7, a TGF-β family-inducible gene, was increased by BDV infection, and the expression was decreased by treatment with A-83-01 or LDN-193189, inhibitors of the TGF-β/activin or BMP pathway, respectively. These results suggest autocrine effects of activin A and BMP7 in C6BV cells. IGFBP-3 expression was also induced by BDV infection; it was below the detection limit in C6 cells. The expression level of IGFBP-3 was decreased by LDN-193189 in C6BV cells, suggesting that endogenous BMP activity is responsible for IGFBP-3 gene induction. Our results reveal the regulatory expression of genes related to the TGF-β family, and the role of the enhanced BMP pathway in modulating cell responses in BDV-infected glial cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  6. MAPK Phosphatase 5 Expression Induced by Influenza and Other RNA Virus Infection Negatively Regulates IRF3 Activation and Type I Interferon Response

    Directory of Open Access Journals (Sweden)

    Sharmy J. James

    2015-03-01

    Full Text Available The type I interferon system is essential for antiviral immune response and is a primary target of viral immune evasion strategies. Here, we show that virus infection induces the expression of MAPK phosphatase 5 (MKP5, a dual-specificity phosphatase (DUSP, in host cells. Mice deficient in MKP5 were resistant to H1N1 influenza infection, which is associated with increased IRF3 activation and type I interferon expression in comparison with WT mice. Increased type I interferon responses were also observed in MKP5-deficient cells and animals upon other RNA virus infection, including vesicular stomatitis virus and sendai virus. These observations were attributed to the ability of MKP5 to interact with and dephosphorylate IRF3. Our study reveals a critical function of a DUSP in negative regulation of IRF3 activity and demonstrates a mechanism by which influenza and other RNA viruses inhibit type I interferon response in the host through MKP5.

  7. Recombinant rabies virus expressing IL-21 enhances immunogenicity through activation of T follicular helper cells and germinal centre B cells.

    Science.gov (United States)

    Zhang, Yajing; Zhou, Ming; Wang, Zhao; Yang, Jie; Li, Mingming; Wang, Kunlun; Cui, Min; Chen, Huanchun; Fu, Zhen F; Zhao, Ling

    2016-12-01

    Previous studies have demonstrated that the lack of interleukin-21 (IL-21) signalling could affect specific antibody induction after rabies vaccination. Here, to further investigate the over-expression of IL-21 on the immunogenicity of rabies virus (RABV), a recombinant RABV expressing murine IL-21, designated LBNSE-IL21, was constructed and evaluated in a mouse model. It was found that in mice immunized with LBNSE-IL21, there was a substantial increase in the number of T follicular helper cells and germinal centre B cells but no enhancement of dendritic cell activation. Furthermore, significantly higher rabies virus-neutralizing antibody (VNA) titres were produced in mice immunized with LBNSE-IL21 than in mice immunized with the parent virus LBNSE in the first six weeks, resulting in higher protection. Together, these results suggest that LBNSE-IL21 can induce a rapid and robust VNA titre, and it has the potential to be developed as a promising rabies vaccine.

  8. Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ.

    Science.gov (United States)

    Barkhouse, Darryll A; Faber, Milosz; Hooper, D Craig

    2015-01-01

    Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    Science.gov (United States)

    Makvandi, Manoochehr; Teimoori, Ali; Neisi, Niloofar; Samarbafzadeh, Alireza

    2016-01-01

    Background The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5α and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results The lengths of subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. PMID:28138375

  10. Analysis of the miRNA expression profile in an Aedes albopictus cell line in response to bluetongue virus infection.

    Science.gov (United States)

    Xing, Shanshan; Du, Junzheng; Gao, Shandian; Tian, Zhancheng; Zheng, Yadong; Liu, Guangyuan; Luo, Jianxun; Yin, Hong

    2016-04-01

    Cellular microRNAs (miRNAs) have been reported to be key regulators of virus-host interactions. Bluetongue virus (BTV) is an insect-borne virus that causes huge economic losses in the livestock industry worldwide. Aedes albopictus cell lines have become powerful and convenient tools for studying BTV-vector interactions. However, the role of miRNAs in A. albopictus cells during BTV infection is not well understood. In this study, we performed a deep sequencing analysis of small RNA libraries of BTV-infected and mock-infected A. albopictus cells, and a total of 11,206,854 and 12,125,274 clean reads were identified, respectively. A differential expression analysis showed that 140 miRNAs, including 15 known and 125 novel miRNAs, were significantly dysregulated after infection, and a total of 414 and 2307 target genes were annotated, respectively. Real-time quantitative reverse transcription-polymerase chain reaction validated the expression patterns of 11 selected miRNAs and their mRNA targets. Functional annotation of the target genes suggested that these target genes were mainly involved in metabolic pathways, oxidative phosphorylation, endocytosis, RNA transport, as well as the FoxO, Hippo, Jak-STAT, and MAPK signaling pathways. This is the first systematic study on the effect of BTV infection on miRNA expression in A. albopictus cells. This investigation provides information concerning the cellular miRNA expression profile in response to BTV infection, and it offers clues for identifying potential candidates for vector-based antiviral strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Expression and Characterization of a Soluble Form of Tomato Spotted Wilt Virus Glycoprotein GN

    OpenAIRE

    Whitfield, Anna E.; Ullman, Diane E.; German, Thomas L

    2004-01-01

    Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, GN and GC, are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play ...

  12. Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Breum, Solvej Østergaard; Riber, Ulla

    2014-01-01

    Background: Major histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads...... to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. Findings: Four SwIV derived peptides were...... subsets indicating multiple specificities. Conclusions: This study describes a timely and cost-effective approach for viral epitope identification in livestock animals. Analysis of T cell subsets showed multiple specificities suggesting SLA-bound epitope recognition of different conformations....

  13. VEGF-Mediated Induction of PRD1-BF1/Blimp1 Expression Sensitizes Tumor Vasculature to Oncolytic Virus Infection.

    Science.gov (United States)

    Arulanandam, Rozanne; Batenchuk, Cory; Angarita, Fernando A; Ottolino-Perry, Kathryn; Cousineau, Sophie; Mottashed, Amelia; Burgess, Emma; Falls, Theresa J; De Silva, Naomi; Tsang, Jovian; Howe, Grant A; Bourgeois-Daigneault, Marie-Claude; Conrad, David P; Daneshmand, Manijeh; Breitbach, Caroline J; Kirn, David H; Raptis, Leda; Sad, Subash; Atkins, Harold; Huh, Michael S; Diallo, Jean-Simon; Lichty, Brian D; Ilkow, Carolina S; Le Boeuf, Fabrice; Addison, Christina L; McCart, J Andrea; Bell, John C

    2015-08-10

    Oncolytic viruses designed to attack malignant cells can in addition infect and destroy tumor vascular endothelial cells. We show here that this expanded tropism of oncolytic vaccinia virus to the endothelial compartment is a consequence of VEGF-mediated suppression of the intrinsic antiviral response. VEGF/VEGFR2 signaling through Erk1/2 and Stat3 leads to upregulation, nuclear localization, and activation of the transcription repressor PRD1-BF1/Blimp1. PRD1-BF1 does not contribute to the mitogenic effects of VEGF, but directly represses genes involved in type I interferon (IFN)-mediated antiviral signaling. In vivo suppression of VEGF signaling diminishes PRD1-BF1/Blimp1 expression in tumor vasculature and inhibits intravenously administered oncolytic vaccinia delivery to and consequent spread within the tumor. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Inhibition of G1P3 expression found in the differential display study on respiratory syncytial virus infection

    Directory of Open Access Journals (Sweden)

    Li Lei

    2008-10-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is the leading viral pathogen associated with bronchiolitis and lower respiratory tract disease in infants and young children worldwide. The respiratory epithelium is the primary initiator of pulmonary inflammation in RSV infections, which cause significant perturbations of global gene expression controlling multiple cellular processes. In this study, differential display reverse transcription polymerase chain reaction amplification was performed to examine mRNA expression in a human alveolar cell line (SPC-A1 infected with RSV. Results Of the 2,500 interpretable bands on denaturing polyacrylamide gels, 40 (1.6% cDNA bands were differentially regulated by RSV, in which 28 (70% appeared to be upregulated and another 12 (30% appeared to be downregulated. Forty of the expressed sequence tags (EST were isolated, and 20 matched homologs in GenBank. RSV infection upregulated the mRNA expression of chemokines CC and CXC and interfered with type α/β interferon-inducible gene expression by upregulation of MG11 and downregulation of G1P3. Conclusion RSV replication could induce widespread changes in gene expression including both positive and negative regulation and play a different role in the down-regulation of IFN-α and up-regulation of IFN-γ inducible gene expression, which suggests that RSV interferes with the innate antiviral response of epithelial cells by multiple mechanisms.

  15. CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2013-12-01

    Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

  16. TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

    Science.gov (United States)

    Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

    2012-01-01

    Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

  17. Conformation-dependent recognition of baculovirus-expressed Epstein-Barr virus gp350 by a panel of monoclonal antibodies.

    Science.gov (United States)

    Zhang, P F; Marcus-Sekura, C J

    1993-10-01

    The Epstein-Barr virus (EBV) major membrane protein, gp350, induces antibodies that neutralize virus infectivity in vitro and is a potential candidate for an EBV vaccine. Full-length EBV gp350 and five protein fragments, encompassing the entire protein sequence, were generated in a baculovirus expression system. The recombinant proteins were analysed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared against native gp350 derived from virus-producing cells and one prepared against an Escherichia coli recombinant protein). All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombinant gp350 in a dot blot immunoassay, but only four of the 14 MAbs reacted with polypeptides expressed by the five subclones, indicating that the full-length protein, but not the protein fragments, was antigenically similar to native gp350. Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-length gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated. PNGase F treatment of the full-length glycosylated gp350 did not eliminate its reactivity with all of the 10 MAbs examined (including the neutralizing MAb) in a dot blot immunoassay; however, denatured glycosylated gp350 lost reactivity with all but four of the 14 MAbs when analysed by either dot blot or Western blot immunoassay. The data suggest that conformational epitopes are more important in recognition of gp350 by this panel of MAbs than glycosylation sites, and that the epitope on gp350 recognized by the neutralizing MAb is conformation- and not glycosylation-dependent.

  18. T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein

    Science.gov (United States)

    Hausmann, Jürgen; Hallensleben, Wiebke; de la Torre, Juan Carlos; Pagenstecher, Axel; Zimmermann, Christine; Pircher, Hanspeter; Staeheli, Peter

    1999-01-01

    Infection of neonates with Borna disease virus (BDV) induces severe meningoencephalitis and neurological disorder in wild-type but not in β2-microglobulin-deficient mice of strain MRL (H-2k). Temporary in vivo depletion of CD8+ T cells delayed BDV-induced disease for several weeks. Depletion of CD4+ T cells had a similar beneficial effect, indicating that the BDV-induced neurological disorder in mice is a CD4+ T cell-dependent immunopathological process that is mediated by CD8+ T cells. Lymphocytes prepared from brains of diseased mice were mainly from the CD8+ T cell subset. They showed up-regulation of activation markers and exerted strong MHC I-restricted cytotoxic activity against target cells expressing the BDV nucleoprotein p40. Infection of B10.BR (H-2k) or congenic C57BL/10 (H-2b) mice resulted in symptomless, lifelong persistence of BDV in the brain. Superinfection with a recombinant vaccinia virus expressing BDV p40 but not with other vaccinia viruses induced severe neurological disease and encephalitis in persistently infected B10.BR mice but not in persistently infected C57BL/10 mice, indicating that the disease-inducing T cell response is restricted to the nucleoprotein of BDV in H-2k mice. Our results demonstrate that the cellular arm of the immune system may ignore the presence of a replicating virus in the central nervous system until proper antigenic stimulation at a peripheral site triggers the antiviral response. PMID:10449769

  19. Clinical significance of spasmolytic polypeptide-expressing metaplasia and intestinal metaplasia in Epstein-Barr virus-associated and Epstein-Barr virus-negative gastric cancer.

    Science.gov (United States)

    Zhang, Yu; Chen, Jian-Ning; Dong, Min; Zhang, Zhi-Gang; Zhang, Yi-Wang; Wu, Jun-Yan; Du, Hong; Li, Hai-Gang; Huang, Yan; Shao, Chun-Kui

    2017-05-01

    Spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM) have been recognized as neoplastic precursors in gastric carcinogenesis. We explored the relationship between SPEM and IM in Epstein-Barr virus-associated (EBVaGC) and Epstein-Barr virus-negative (EBVnGC) gastric cancer. Sixty-four EBVaGC and one hundred and fifty-four EBVnGC patients were included. EBV positivity was identified using Epstein-Barr virus-encoded RNA-1 in situ hybridization. SPEM was subclassified into absent, early, and advanced SPEM. Acute and chronic inflammation was graded as absent, mild, moderate, and marked. Univariate and multivariate logistic regression analyses were conducted to analyze the correlation between SPEM, IM, and inflammation. Our study revealed that SPEM was detected in 87.5% EBVaGC and 85.1% EBVnGC patients. Distribution of patients according to the SPEM classification was significantly different between EBVaGC and EBVnGC groups (P=.038). IM was observed less frequently in EBVaGC when compared with EBVnGC patients (P<.001). No difference was observed between EBVaGC and EBVnGC in the levels of acute and chronic inflammation. A positive correlation between IM and SPEM status was observed in both EBVaGC and EBVnGC patients. Furthermore, advanced SPEM was an independent influential factor to IM in EBVnGC (P=.013). In conclusion, SPEM was associated with both EBVaGC and EBVnGC more frequently than IM. Moreover, advanced SPEM had a stronger association with IM than early SPEM in EBVnGC. These results suggest that identification of SPEM should be used as a high-risk indicator for detecting early gastric carcinoma, and should be brought to the attention of pathologists and clinicians. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Influence of white spot syndrome virus infection on hepatopancreas gene expression of `Huanghai No. 2' shrimp ( Fenneropenaeus chinensis)

    Science.gov (United States)

    Meng, Xianhong; Shi, Xiaoli; Kong, Jie; Luan, Sheng; Luo, Kun; Cao, Baoxiang; Liu, Ning; Lu, Xia; Li, Xupeng; Deng, Kangyu; Cao, Jiawang; Zhang, Yingxue; Zhang, Hengheng

    2017-10-01

    To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of `Huanghai No. 2' ( Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48-96 h), peak (168-192 h) and late (264-288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.

  1. Resistance to simian immunodeficiency virus low dose rectal challenge is associated with higher constitutive TRIM5α expression in PBMC.

    Science.gov (United States)

    Aamer, Hadega A; Rajakumar, Premeela; Nyaundi, Julia; Murphey-Corb, Michael

    2014-05-23

    At least six host-encoded restriction factors (RFs), APOBEC3G, TRIM5α, tetherin, SAMHD1, schlafen 11, and Mx2 have now been shown to inhibit HIV and/or SIV replication in vitro. To determine their role in vivo in the resistance of macaques to mucosally-acquired SIV, we quantified both pre-exposure (basal) and post-exposure mRNA levels of these RFs, Mx1, and IFNγ in PBMC, lymph nodes, and duodenum of rhesus macaques undergoing weekly low dose rectal exposures to the primary isolate, SIV/DeltaB670. Repetitive challenge divided the monkeys into two groups with respect to their susceptibility to infection: highly susceptible (2-3 challenges, 5 monkeys) and poorly susceptible (≥6 challenges, 3 monkeys). Basal RF and Mx1 expression varied among the three tissues examined, with the lowest expression generally detected in duodenal tissues, and the highest observed in PBMC. The one exception was A3G whose basal expression was greatest in lymph nodes. Importantly, significantly higher basal expression of TRIM5α and Mx1 was observed in PBMC of animals more resistant to mucosal infection. Moreover, individual TRIM5α levels were stable throughout a year prior to infection. Post-exposure induction of these genes was also observed after virus appearance in plasma, with elevated levels in PBMC and duodenum transiently occurring 7-10 days post infection. They did not appear to have an effect on control of viremia. Interestingly, minimal to no induction was observed in the resistant animal that became an elite controller. These results suggest that constitutively expressed TRIM5α appears to play a greater role in restricting mucosal transmission of SIV than that associated with type I interferon induction following virus entry. Surprisingly, this association was not observed with the other RFs. The higher basal expression of TRIM5α observed in PBMC than in duodenal tissues emphasizes the understated role of the second barrier to systemic infection involving the transport of

  2. Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine.

    Directory of Open Access Journals (Sweden)

    Zhiyong Li

    Full Text Available BACKGROUND: Foot-and-mouth disease (FMD is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD(50 (50% bovine protective dose test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD(50 per dose. CONCLUSION: The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.

  3. Expression and immunological studies of classical swine fever virus glycoprotein E2 in the bi-cistronic baculovirus/larvae expression system.

    Science.gov (United States)

    Wu, Chi-Ming; Hsuan, Shih-Ling; Chen, Zeng-Weng; Jinn, Tzyy-Rong; Huang, Chienjin; Liao, Jiunn-Wang; Chen, Ter-Hsin; Liao, Chih-Ming; Lee, Wei-Cheng; Wu, Tzong-Yuan; Chien, Maw-Sheng

    2010-01-01

    To develop an economical, easy technique for producing recombinant E2 glycoprotein (rE2) of classical swine fever virus (CSFV) as a candidate immunogen, a bi-cistronic baculovirus/larvae expression vector was constructed using p10 promoter, an internal ribosome entry site, and the gfp gene. Trichoplusia ni larvae were successfully infected with the occluded recombinant baculovirus via feed, and the characteristics of rE2 were confirmed by immunoblot and glycosylation stain. rE2 at a concentration of 0.6-0.8 mg/ml without degradation was obtained from hemolymphs of infected larvae that emitted high levels of green fluorescence. Immunization assays indicated that mice and piglets immunized with rE2-containing hemolymph elicited high titers of anti-CSFV E2 antibodies with virus-neutralizing activity. This is the first study to indicate that baculovirus/T. ni larvae-expressed rE2 can be served as a vaccine candidate. This system provides an economical alternative for the production of vaccine components in the veterinary industry.

  4. Production and diagnostic application of a purified, E. coli-expressed, serological-specific chicken anaemia virus antigen VP3.

    Science.gov (United States)

    Lee, M-S; Chou, Y-M; Lien, Y-Y; Lin, M-K; Chang, W-T; Lee, H-Z; Lee, M-S; Lai, G-H; Chen, H-J; Huang, C-H; Lin, W-H

    2011-06-01

    The aim of this study was to evaluate the production of chicken anaemia virus VP3 protein in different Escherichia coli strains and to address the diagnostic application of purified E. coli-expressed VP3 protein for the detection of chicken anaemia virus (CAV) infection and the development of an ELISA kit. Three E. coli strains, BL21, BL21 codonplus RP and BL21 pLysS, each harbouring a VP3 protein expressing plasmid, were investigated after induction to produce recombinant VP3 protein. After isopropyl-β-D-thiogalactoside (IPTG) induction, VP3 protein was successfully expressed in all three E. coli strains. The BL21 pLysS strain gave the best performance in terms of protein productivity and growth profile. In addition, the optimal culture temperature and IPTG concentration were found to be 0.25 mM and 20 °C, respectively. Using Ni-NTA-purified VP3 protein as an ELISA coating antigen, the purified VP3 was shown to be highly antigenic and able to discriminate sera from chickens infected with CAV from those that were uninfected during an evaluation of CAV infection serodiagnosis. A VP3-based ELISA demonstrated 100% (6/6 x 100%) specificity and sensitivities of 91.3% (21/23 x 100%) and 82.6% (19/23 x 100%) using cut-off values of the mean plus 2 SD and the mean plus 3 SD, respectively. © 2011 Blackwell Verlag GmbH.

  5. Genotyping, levels of expression and physical status of human papilloma virus in oropharyngeal squamous cell carcinoma among Colombian patients.

    Science.gov (United States)

    Erira, Alveiro; Motta, Leidy Angélica; Chala, Andrés; Moreno, Andrey; Gamboa, Fredy; García, Dabeiba Adriana

    2015-10-23

    One of the risk factors for squamous cell oropharyngeal carcinoma is infection with the human papilloma virus (HPV), with prevalences that vary depending on the geographical region.  To identify the most frequent HPV viral types in oropharyngeal cancer, the levels of expression and the physical condition of the viral genome.  Forty-six patients were included in the study from among those attending head and neck surgical services in the cities of Bogotá, Manizales and Bucaramanga. In the histopathological report all study samples were characterized as oropharyngeal squamous cell carcinoma. DNA extraction was subsequently performed for HPV genotyping and to determine the physical state of the viral genome, as well as RNA to determine viral transcripts using real-time PCR.  HPV prevalence in tumors was 21.74% (n=10) and the most common viral type was HPV-16 (nine cases). Viral expression for HPV-16 was low (one of 11 copies) and the predominant physical state of the virus was mixed (eight cases), with disruption observed at the E1 - E2 binding site (2525 - 3720 nucleotides).  The prevalence of HPV associated with oropharyngeal carcinoma among the Colombian study population was 21.7%, which is relatively low. The most frequent viral type was HPV-16, found in a mixed form and with low expression of E7, possibly indicating a poor prognosis for these patients.

  6. Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis

    Energy Technology Data Exchange (ETDEWEB)

    Baczko, K.; Liebert, U.G.; Billeter, M.; Cattaneo, R.; Budka, H.; Ter Meulen, V.

    1986-08-01

    The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the bran sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.

  7. Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

    Science.gov (United States)

    Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

    2017-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

  8. Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.

    Science.gov (United States)

    Wang, A P; Sun, H C; Wang, J Y; Wang, Y J; Yuan, W F

    2008-04-01

    Human tissue kallikrein (hK1) plays an important role in regulation of blood pressure, electrolyte and glucose transport, and renal function. To evaluate the feasibility of viral vector-mediated expression of recombinant human tissue kallikrein (rhK1) in the egg white of laying hens, human tissue kallikrein gene (hKLK1) cDNA-expression cassette was subcloned into avian adeno-associated virus (AAAV) transfer vector pAITR and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper. The recombinant viral particles with a typical AAAV morphology and relatively high titer were generated and identified by PCR and electron microscopy. After 1 intravenous injection of each laying hen with 2 x 10(10) viral particles, oviduct-specific expression of hKLK1 cDNA was demonstrated by reverse transcription-PCR. Secretion of rhK1 into the egg white was detected by enzymatic assay from d 2, reaching the highest level of 107 U/mL in wk 3, and lasted for more than 6 wk after injection. Western blotting showed that the oviduct-expressed rhK1 had the same molecular mass with the natural enzyme. These data suggest that rAAAV can mediate high level and long-lasting transgene expression in oviduct cells, and the established expression system is useful for production of other recombinant proteins.

  9. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang

    2013-01-01

    Full Text Available Pectinesterase inhibitor (PEI isolated from jelly fig (Ficus awkeotsang Makino is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg. Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B and integrated (Huh7 HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  10. Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9

    Directory of Open Access Journals (Sweden)

    Jason L Picconi

    2014-01-01

    Full Text Available Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV-enhanced green fluorescent protein (eGFP vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

  11. Hepatitis B Virus X Upregulates HuR Protein Level to Stabilize HER2 Expression in Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Chao-Ming Hung

    2014-01-01

    Full Text Available Hepatitis B virus- (HBV- associated hepatocellular carcinoma (HCC is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2 is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC.

  12. MHC Expression on Spleen Lymphocyte Subsets in Genetically Resistant and Susceptible Chickens Infected with Marek's Disease Virus

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Bøving, Mette K.; Handberg, Kurt

    2009-01-01

    Resistance and susceptibility to Marek's disease (MD) are strongly influenced by the chicken major histocompatibility complex (MHC). In this study, splenic lymphocytes from MD-resistant and MD-susceptible chickens of three MHC genotypes (B21/B21, B19/B21, and B19/B19) were analyzed by flow...... genotypes were subjected to infection with MD virus (GA strain) and spleen samples from infected as well as MHC-matched negative controls were analyzed at 1, 4, and 8 wk post-infection (p.i.). It was observed that MDV induced an increase in MHC class I expression late in the infection. Thus, MHC class I...

  13. Replication of different clones of human immunodeficiency virus type 1 in primary fetal human astrocytes: enhancement of viral gene expression by Nef.

    Science.gov (United States)

    Bencheikh, M; Bentsman, G; Sarkissian, N; Canki, M; Volsky, D J

    1999-04-01

    Dementia is a common complication of AIDS which is associated with human immunodeficiency virus type 1 (HIV-1) infection of brain macrophages and microglia. Recent studies have shown that astrocytes are also infected in the brain but HIV-1 replication in these cells is restricted. To determine virus specificity of this restriction we tested the expression of 15 HIV-1 molecular clones in primary human fetal astrocytes by infection and DNA transfection. Infection with cell-free viruses was poorly productive and revealed no clone-specific differences. In contrast, transfected cells produced transiently high levels of HIV-1 p24 core antigen, up to 50 nanograms per ml culture supernatant, and nanogram levels of p24 were detected 3-4 weeks after transfection of some viral clones. The average peak expression of HIV-1 in astrocytes varied as a function of viral clone used by a factor of 15 but the differences and the subsequent virus spread did not correlate with the tropism of the viral clones to T cells or m