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Sample records for prrsv nonstructural protein

  1. Immune Response of Multiparous Hyper-Immunized Sows against Peptides from Non-Structural and Structural Proteins of PRRSV

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    Edgar Rascón-Castelo

    2015-11-01

    Full Text Available The purpose of this study was to evaluate the humoral and cellular responses of commercial multiparous and hyper-immunized sows against peptides from non-structural (nsp and structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV. We selected sows with different numbers of parities from a commercial farm. Management practices on this farm include the use of the MLV commercial vaccine four times per year, plus two vaccinations during the acclimation period. The humoral response was evaluated via the antibody recognition of peptides from nsp and structural proteins, and the cellular response was assessed by measuring the frequency of peptide and PRRSV-specific IFN-gamma-secreting cells (IFNγ-SC. Our results show that sows with six parities have more antibodies against peptides from structural proteins than against peptides from nsp. The analysis of the cellular response revealed that the number of immunizations did not affect the frequency of IFNγ-SC and that the response was stronger against peptides from structural proteins (M protein than against nsp (nsp2. In summary, these results demonstrate that multiparous, hyper-immunized sows have a stronger immune humoral response to PRRSV structural peptides than nsp, but no differences in IFNγ-SC against the same peptides were observed.

  2. The non-structural protein 5 and matrix protein are antigenic targets of T cell immunity to genotype 1 porcine reproductive and respiratory syndrome viruses

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    Helen eMokhtar

    2016-02-01

    Full Text Available The porcine reproductive and respiratory syndrome virus (PRRSV is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focussed on envelope glycoproteins to target virus-neutralising antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress towards market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralising antibodies, it has been proposed that T cell mediated immunity plays a key role. We therefore hypothesised that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely-related (subtype 1 or divergent (subtype 3 PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5, and to a lesser extent, the matrix (M protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by co-expression of TNF-α and mobilisation of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved amongst strains of both PRRSV genotypes. Thus M and NSP5 represent attractive vaccine candidate T cell antigens which should be evaluated further in the context of PRRSV vaccine development.

  3. Role of nonstructural protein NS2A in flavivirus assembly

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    Leung, J.Y.; Pijlman, G.P.; Kondratieva, N.; Hyde, J.; Mackenzie, J.M.; Khromykh, A.A.

    2008-01-01

    Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part

  4. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

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    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG

    2016-06-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  5. MAVS protein is attenuated by rotavirus nonstructural protein 1.

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    Satabdi Nandi

    Full Text Available Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS, which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1 which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.

  6. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    International Nuclear Information System (INIS)

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

  7. Cleft analysis of Zika virus non-structural protein 1

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    Somsri Wiwanitkit; Viroj Wiwanitkit

    2017-01-01

    The non-structural protein 1 is an important molecule of the viruses in flavivirus group including to Zika virus. Recently, the NS1 of Zika virus was discovered. There is still no complete information of the molecular interaction of NS1 of Zika virus which can be the clue for explanation for its pathogenesis and further drug search. Here the authors report the cleft analysis of NS1 of Zika virus and the result can be useful for future development of good diagnostic tool and antiviral drug finding for management of Zika virus.

  8. Cleft analysis of Zika virus non-structural protein 1

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    Somsri Wiwanitkit

    2017-08-01

    Full Text Available The non-structural protein 1 is an important molecule of the viruses in flavivirus group including to Zika virus. Recently, the NS1 of Zika virus was discovered. There is still no complete information of the molecular interaction of NS1 of Zika virus which can be the clue for explanation for its pathogenesis and further drug search. Here the authors report the cleft analysis of NS1 of Zika virus and the result can be useful for future development of good diagnostic tool and antiviral drug finding for management of Zika virus.

  9. Cleft analysis of Zika virus non-structural protein 1

    OpenAIRE

    Somsri Wiwanitkit; Viroj Wiwanitkit

    2017-01-01

    The non-structural protein 1 is an important molecule of the viruses in flavivirus group including to Zika virus. Recently, the NS1 of Zika virus was discovered. There is still no complete information of the molecular interaction of NS1 of Zika virus which can be the clue for explanation for its pathogenesis and further drug search. Here the authors report the cleft analysis of NS1 of Zika virus and the result can be useful for future development of good diagnostic tool and antiviral drug fin...

  10. Innate Immune Evasion Mediated by Flaviviridae Non-Structural Proteins.

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    Chen, Shun; Wu, Zhen; Wang, Mingshu; Cheng, Anchun

    2017-10-07

    Flaviviridae-caused diseases are a critical, emerging public health problem worldwide. Flaviviridae infections usually cause severe, acute or chronic diseases, such as liver damage and liver cancer resulting from a hepatitis C virus (HCV) infection and high fever and shock caused by yellow fever. Many researchers worldwide are investigating the mechanisms by which Flaviviridae cause severe diseases. Flaviviridae can interfere with the host's innate immunity to achieve their purpose of proliferation. For instance, dengue virus (DENV) NS2A, NS2B3, NS4A, NS4B and NS5; HCV NS2, NS3, NS3/4A, NS4B and NS5A; and West Nile virus (WNV) NS1 and NS4B proteins are involved in immune evasion. This review discusses the interplay between viral non-structural Flaviviridae proteins and relevant host proteins, which leads to the suppression of the host's innate antiviral immunity.

  11. Functional investigation of grass carp reovirus nonstructural protein NS80

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    Shao Ling

    2011-04-01

    Full Text Available Abstract Background Grass Carp Reovirus (GCRV, a highly virulent agent of aquatic animals, has an eleven segmented dsRNA genome encased in a multilayered capsid shell, which encodes twelve proteins including seven structural proteins (VP1-VP7, and five nonstructural proteins (NS80, NS38, NS31, NS26, and NS16. It has been suggested that the protein NS80 plays an important role in the viral replication cycle that is similar to that of its homologous protein μNS in the genus of Orthoreovirus. Results As a step to understanding the basis of the part played by NS80 in GCRV replication and particle assembly, we used the yeast two-hybrid (Y2H system to identify NS80 interactions with proteins NS38, VP4, and VP6 as well as NS80 and NS38 self-interactions, while no interactions appeared in the four protein pairs NS38-VP4, NS38-VP6, VP4-VP4, and VP4-VP6. Bioinformatic analyses of NS80 with its corresponding proteins were performed with all currently available homologous protein sequences in ARVs (avian reoviruses and MRVs (mammalian reoviruses to predict further potential functional domains of NS80 that are related to VFLS (viral factory-like structures formation and other roles in viral replication. Two conserved regions spanning from aa (amino acid residues of 388 to 433, and 562 to 580 were discovered in this study. The second conserved region with corresponding conserved residues Tyr565, His569, Cys571, Asn573, and Glu576 located between the two coiled-coils regions (aa ~513-550 and aa ~615-690 in carboxyl-proximal terminus were supposed to be essential to form VFLS, so that aa residues ranging from 513 to 742 of NS80 was inferred to be the smallest region that is necessary for forming VFLS. The function of the first conserved region including Ala395, Gly419, Asp421, Pro422, Leu438, and Leu443 residues is unclear, but one-third of the amino-terminal region might be species specific, dominating interactions with other viral components. Conclusions Our

  12. Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

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    Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

    2014-08-01

    The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites

  13. Membrane alterations induced by nonstructural proteins of human norovirus.

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    Sylvie Y Doerflinger

    2017-10-01

    Full Text Available Human noroviruses (huNoV are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4 variants. The viral nonstructural (NS proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV. Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER which included single membrane vesicles (SMVs, double membrane vesicles (DMVs and multi membrane vesicles (MMVs. In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and

  14. Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses

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    Han, Mingyuan; Kim, Chi Yong [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802 (United States); Rowland, Raymond R.R.; Fang, Ying [Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506 (United States); Kim, Daewoo [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802 (United States)

    2014-06-15

    Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV–nsp1 and LDV–nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV–nsp1 remained uncleaved. SHFV–nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV–nsp1αβ and SHFV–nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV–nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV–nsp1β, LDV–nsp1β, EAV–nsp1, and SHFV–nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV–nsp1α, CBP degradation was evident in cells expressing LDV–nsp1α and SHFV–nsp1γ, but no such degradation was observed for EAV–nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ

  15. Structure and non-structure of centrosomal proteins.

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    Dos Santos, Helena G; Abia, David; Janowski, Robert; Mortuza, Gulnahar; Bertero, Michela G; Boutin, Maïlys; Guarín, Nayibe; Méndez-Giraldez, Raúl; Nuñez, Alfonso; Pedrero, Juan G; Redondo, Pilar; Sanz, María; Speroni, Silvia; Teichert, Florian; Bruix, Marta; Carazo, José M; Gonzalez, Cayetano; Reina, José; Valpuesta, José M; Vernos, Isabelle; Zabala, Juan C; Montoya, Guillermo; Coll, Miquel; Bastolla, Ugo; Serrano, Luis

    2013-01-01

    Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

  16. Cleft analysis of Zika virus non-structural protein 1

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    Somsri Wiwanitkit; Viroj Wiwanitkit

    2017-01-01

    The non-strctural protein 1 is an important molecule of the viruses in flavivirus group including to Zika virus. Recently, the NS1 of Zika virus was discovered.There is still no complete information of the molecular interaction of NS1 of Zika virus which can be the clue for explanation for its pathogenesis and further drug search. Here the authors report the cleft analysis of NS1 of Zika virus and the result can be useful for future development of good diagnostic tool and antiviral drug finding for management of Zika virus.

  17. Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA

    International Nuclear Information System (INIS)

    Burnham, Andrew J.; Gong, Lei; Hardy, Richard W.

    2007-01-01

    Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed

  18. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

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    Boyce Mark

    2012-08-01

    Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

  19. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

    Science.gov (United States)

    Boyce, Mark; Celma, Cristina C P; Roy, Polly

    2012-08-29

    Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

  20. Identification and characterization of a novel non-structural protein of bluetongue virus.

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    Maxime Ratinier

    2011-12-01

    Full Text Available Bluetongue virus (BTV is the causative agent of a major disease of livestock (bluetongue. For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4 encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

  1. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    International Nuclear Information System (INIS)

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-01-01

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  2. [Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

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    Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

    2015-11-01

    Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

  3. Identification of a major non-structural protein in the nuclei of Rift Valley fever virus-infected cells.

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    Struthers, J K; Swanepoel, R

    1982-06-01

    A non-structural protein of mol. wt. 34 X 10(3) was demonstrated in the nuclei of Rift Valley fever virus-infected Vero cells by SDS-polyacrylamide gel electro-phoresis. The protein appears to correspond to the virus-induced antigen demonstrated by indirect immunofluorescence in intranuclear inclusions.

  4. The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

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    URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

    2015-01-01

    The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

  5. Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

    International Nuclear Information System (INIS)

    Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee; Afanasiev, Boris; Kittayapong, Pattamaporn; Carlson, Jonathan; Corsini, Joe

    2008-01-01

    Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny

  6. Non-Canonical Roles of Dengue Virus Non-Structural Proteins

    Directory of Open Access Journals (Sweden)

    Julianna D. Zeidler

    2017-03-01

    Full Text Available The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV non-structural proteins (NSPs, which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection.

  7. Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

    Science.gov (United States)

    Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

    2017-01-01

    Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

    Science.gov (United States)

    Ribaudo, Michael; Barik, Sailen

    2017-11-06

    Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

  9. Polypeptide structure and encoding location of the adenovirus serotype 2 late, nonstructural 33K protein

    International Nuclear Information System (INIS)

    Oosterom-Dragon, E.A.; Anderson, C.W.

    1983-01-01

    Radiochemical microsequence analysis of selected tryptic peptides of the adenovirus type 2 33K nonstructural protein has revealed the precise region of the genomic nucleotide sequence that encodes this protein. The initiation codon for the 33K protein lies 606 nucleotides to the right of the EcoRI restriction site at 70.7 map units and 281 nucleotides to the left of the postulated carboxyterminal codon of the adenovirus 100K protein. The coding regions for these two proteins thus overlap; however, the 33K protein is derived from the +1 frame with respect to the postulated 100K reading frame. Our results contradict an earlier published report suggesting that these two proteins share extensive amino acid sequence homology. The published nucleotide sequence of the Ad2 EcoRI-F fragment (70.7 to 75.9 map units) cannot accomodate in a single reading frame the peptide sequences of the 33K protein that we have determined. Sequence analysis of DNA fragments derived from virus has confirmed the published nucleotide sequence in all critical regions with respect to the coding region for the 33K protein. Consequently, our data are only consistent with the existence of an mRNA splice within the coding for 33K. Consensus donor and acceptor splice sequences have been located that would predict the removal of 202 nucleotides from the transcripts for the 33K protein. Removal of these nucleotides would explain the structure of a peptide that cannot otherwise be directly encoded by the EcoRI-F fragment. Identification of the precise splice points by peptide sequencing has permitted a prediction of the complete amino acid sequence for the 33K protein

  10. 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

    Science.gov (United States)

    Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

    2017-07-04

    Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

  11. O'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Kali D Saxton-Shaw

    Full Text Available O'nyong nyong virus (ONNV and Chikungunya virus (CHIKV are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3, was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3 replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  13. Nuclear export signal of PRRSV NSP1α is necessary for type I IFN inhibition

    International Nuclear Information System (INIS)

    Chen, Zhi; Liu, Shaoning; Sun, Wenbo; Chen, Lei; Yoo, Dongwan; Li, Feng; Ren, Sufang; Guo, Lihui; Cong, Xiaoyan; Li, Jun; Zhou, Shun; Wu, Jiaqiang

    2016-01-01

    The nonstructural protein 1α (NSP1α) of porcine reproductive and respiratory syndrome virus (PRRSV) is a nucleo-cytoplasmic protein that suppresses the production of type I interferon (IFN). In this study, we investigated the relationship between the subcellular distribution of NSP1α and its inhibition of type I IFN. NSP1α was found to contain the classical nuclear export signal (NES) and NSP1α nuclear export was CRM-1-mediated. NSP1α was shuttling between the nucleus and cytoplasm. We also showed that the nuclear export of NSP1α was necessary for its ability for type I IFN inhibition. NSP1α was also found to interact with CBP, which implies a possible mechanism of CBP degradation by NSP1α. Taken together, our results describe a novel mechanism of PRRSV NSP1α for type I IFN inhibition and suppression of the host innate antiviral response. - Highlights: •NSP1α contains the NES and NSP1α nuclear export was CRM-1-mediated. •NSP1α was shuttling between the nucleus and cytoplasm continuously. •The nuclear export of NSP1α was necessary for its ability for type I IFN inhibition. •NSP1α interacts with CBP, which implies the mechanism of CBP degradation by NSP1α.

  14. Nuclear export signal of PRRSV NSP1α is necessary for type I IFN inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi [Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Sangyuan Road No. 8, Jinan 250100 (China); Liu, Shaoning [Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Sangyuan Road No. 8, Jinan 250100 (China); Shandong Institute of Veterinary Drug Quality Inspection, Shandong Key Laboratory for Quality Safety Monitoring and Risk Assessment of Animal Products, Huaicun Street No. 68, Jinan 250722, Shandong Province (China); Sun, Wenbo; Chen, Lei [Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Sangyuan Road No. 8, Jinan 250100 (China); Yoo, Dongwan [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Feng [Department of Biology and Microbiology, Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD 57007 (United States); Ren, Sufang; Guo, Lihui; Cong, Xiaoyan; Li, Jun [Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Sangyuan Road No. 8, Jinan 250100 (China); Zhou, Shun [College of marine science and engineering, Qingdao Agricultural University, Changcheng Road No. 700, Qingdao 266109 (China); Wu, Jiaqiang, E-mail: wujiaqiang2000@sina.com [Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Sangyuan Road No. 8, Jinan 250100 (China); and others

    2016-12-15

    The nonstructural protein 1α (NSP1α) of porcine reproductive and respiratory syndrome virus (PRRSV) is a nucleo-cytoplasmic protein that suppresses the production of type I interferon (IFN). In this study, we investigated the relationship between the subcellular distribution of NSP1α and its inhibition of type I IFN. NSP1α was found to contain the classical nuclear export signal (NES) and NSP1α nuclear export was CRM-1-mediated. NSP1α was shuttling between the nucleus and cytoplasm. We also showed that the nuclear export of NSP1α was necessary for its ability for type I IFN inhibition. NSP1α was also found to interact with CBP, which implies a possible mechanism of CBP degradation by NSP1α. Taken together, our results describe a novel mechanism of PRRSV NSP1α for type I IFN inhibition and suppression of the host innate antiviral response. - Highlights: •NSP1α contains the NES and NSP1α nuclear export was CRM-1-mediated. •NSP1α was shuttling between the nucleus and cytoplasm continuously. •The nuclear export of NSP1α was necessary for its ability for type I IFN inhibition. •NSP1α interacts with CBP, which implies the mechanism of CBP degradation by NSP1α.

  15. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  16. Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

    Directory of Open Access Journals (Sweden)

    Qin Lan

    2011-12-01

    Full Text Available Abstract Background The nonstructural protein 1 (NSP1 of rotavirus has been reported to block interferon (IFN signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs and (or the β-transducin repeat containing protein (β-TrCP. However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. Results The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I, but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS, indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. Conclusions Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

  17. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A.

    Directory of Open Access Journals (Sweden)

    Margarita Zayas

    2016-01-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein (NS5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI and two intrinsically disordered domains (DII and DIII interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2. We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i SC-dependent recruitment of replication complexes to core protein and (ii BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

  18. Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

    Energy Technology Data Exchange (ETDEWEB)

    Khunchai, Sasiprapa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Junking, Mutita [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Suttitheptumrong, Aroonroong; Yasamut, Umpa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Sawasdee, Nunghathai [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Morchang, Atthapan [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Chaowalit, Prapaipit [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); and others

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

  19. Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

    International Nuclear Information System (INIS)

    Khunchai, Sasiprapa; Junking, Mutita; Suttitheptumrong, Aroonroong; Yasamut, Umpa; Sawasdee, Nunghathai; Netsawang, Janjuree; Morchang, Atthapan; Chaowalit, Prapaipit; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2012-01-01

    Highlights: ► For the first time how DENV NS5 increases RANTES production. ► DENV NS5 physically interacts with human Daxx. ► Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called ‘cytokine storm’, is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

  20. Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus.

    Science.gov (United States)

    Drappier, Melissa; Opperdoes, Fred R; Michiels, Thomas

    2017-07-15

    Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV. IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L

  1. Structure and Function of the Non-Structural Protein of Dengue Virus and its Applications in Antiviral Therapy.

    Science.gov (United States)

    Xie, Qian; Zhang, Bao; Yu, JianHai; Wu, Qinghua; Yang, Fangji; Cao, Hong; Zhao, Wei

    2017-01-01

    Dengue fever, a type of global and tropical infectious disease, and its prevention has become a challenging issue worldwide. Antibody-dependent enhancement effects and the virus pathogenic mechanism have not yet been fully elucidated, hindering the development of dengue fever prevention and suitable drug treatment. There is currently no specific prevention and therapy in clinical trials, however, in recent years, studies have focused on the pathogenesis and treatment of dengue. Research focusing on dengue virus nonstructural protein in special drugs for the prevention and control of dengue fever is a new progress leading to improved understanding regarding the prevention and control of dengue fever and suitable drugs for the treatment. The main challenges regarding the structure of dengue virus nonstructural protein and the drugs for antiviral therapy are summarized in this paper.

  2. The Role of Interferon Antagonist, Non-Structural Proteins in the Pathogenesis and Emergence of Arboviruses

    Directory of Open Access Journals (Sweden)

    Samantha S. Soldan

    2011-06-01

    Full Text Available A myriad of factors favor the emergence and re-emergence of arthropod-borne viruses (arboviruses, including migration, climate change, intensified livestock production, an increasing volume of international trade and transportation, and changes to ecosystems (e.g., deforestation and loss of biodiversity. Consequently, arboviruses are distributed worldwide and represent over 30% of all emerging infectious diseases identified in the past decade. Although some arboviral infections go undetected or are associated with mild, flu-like symptoms, many are important human and veterinary pathogens causing serious illnesses such as arthritis, gastroenteritis, encephalitis and hemorrhagic fever and devastating economic loss as a consequence of lost productivity and high mortality rates among livestock. One of the most consistent molecular features of emerging arboviruses, in addition to their near exclusive use of RNA genomes, is the inclusion of viral, non-structural proteins that act as interferon antagonists. In this review, we describe these interferon antagonists and common strategies that arboviruses use to counter the host innate immune response. In addition, we discuss the complex interplay between host factors and viral determinants that are associated with virus emergence and re-emergence, and identify potential targets for vaccine and anti-viral therapies.

  3. Identification of linear B-cell epitopes on goose parvovirus non-structural protein.

    Science.gov (United States)

    Yu, Tian-Fei; Ma, Bo; Wang, Jun-Wei

    2016-10-15

    Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. Copyright © 2016. Published by Elsevier B.V.

  4. Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

    Science.gov (United States)

    Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

    2016-08-01

    Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection.

  5. Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization*

    Science.gov (United States)

    Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.

    2013-01-01

    The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669

  6. Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization.

    Science.gov (United States)

    Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D

    2013-08-02

    The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.

  7. Genetic Evidence for an Interferon-Antagonistic Function of Rift Valley Fever Virus Nonstructural Protein NSs

    Science.gov (United States)

    Bouloy, Michèle; Janzen, Christian; Vialat, Pierre; Khun, Huot; Pavlovic, Jovan; Huerre, Michel; Haller, Otto

    2001-01-01

    Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR−/− mice lacking the alpha/beta interferon (IFN-α/β) receptor but remained attenuated in IFN-γ receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-α/β production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-α/β and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-α/β production, we infected susceptible IFNAR−/− mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-α/β production. These results demonstrate that the ability of RVFV to inhibit IFN-α/β production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist. PMID:11152510

  8. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

    International Nuclear Information System (INIS)

    Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

    2007-01-01

    Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication

  9. Pharmacoinformatics approach for investigation of alternative potential hepatitis C virus nonstructural protein 5B inhibitors

    Directory of Open Access Journals (Sweden)

    Mirza MU

    2015-03-01

    Full Text Available Muhammad Usman Mirza,1 Noor-Ul-Huda Ghori,2 Nazia Ikram,3 Abdur Rehman Adil,4 Sadia Manzoor3 1Centre for Research in Molecular Medicine (CRiMM, The University of Lahore, Lahore, 2Atta-ur-Rehman School of Applied Biosciences (ASAB, National University of Science and Technology, Islamabad, 3Institute of Molecular Biology and Biotechnology (IMBB, The University of Lahore, Lahore, Pakistan; 4Centre for Excellence in Molecular Biology (CEMB, The University of Punjab, Lahore, Pakistan Abstract: Hepatitis C virus (HCV is one of the major viruses affecting the world today. It is a highly variable virus, having a rapid reproduction and evolution rate. The variability of genomes is due to hasty replication catalyzed by nonstructural protein 5B (NS5B which is also a potential target site for the development of anti-HCV agents. Recently, the US Food and Drug Administration approved sofosbuvir as a novel oral NS5B inhibitor for the treatment of HCV. Unfortunately, it is much highlighted for its pricing issues. Hence, there is an urgent need to scrutinize alternate therapies against HCV that are available at affordable price and do not have associated side effects. Such a need is crucial especially in underdeveloped countries. The search for various new bioactive compounds from plants is a key part of pharmaceutical research. In the current study, we applied a pharmacoinformatics-based approach for the identification of active plant-derived compounds against NS5B. The results were compared to docking results of sofosbuvir. The lead compounds with high-binding ligands were further analyzed for pharmacokinetic and pharmacodynamic parameters based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET profile. The results showed the potential alternative lead compounds that can be developed into commercial drugs having high binding energy and promising ADMET properties. Keywords: hepatitis C, NS5B inhibitors, molecular docking, Auto

  10. Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

    Science.gov (United States)

    Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan; Vitour, Damien

    2017-01-01

    Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this

  11. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  12. Sequence, structure and function relationships in flaviviruses as assessed by evolutive aspects of its conserved non-structural protein domains.

    Science.gov (United States)

    da Fonseca, Néli José; Lima Afonso, Marcelo Querino; Pedersolli, Natan Gonçalves; de Oliveira, Lucas Carrijo; Andrade, Dhiego Souto; Bleicher, Lucas

    2017-10-28

    Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction.

    Science.gov (United States)

    Zhou, Ao; Li, Shuaifeng; Khan, Faheem Ahmed; Zhang, Shujun

    2016-01-01

    Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex.

  14. Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins.

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    Pietro Scaturro

    Full Text Available Non-structural protein 1 (NS1 is one of the most enigmatic proteins of the Dengue virus (DENV, playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E and precursor Membrane (prM. Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

  15. Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release

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    Fei Xiao

    2018-03-01

    Full Text Available Hepatitis C virus (HCV spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2 protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.

  16. Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

    Science.gov (United States)

    Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

    2017-11-01

    The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

  17. Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.; Dinnon, Kenneth H.; Leist, Sarah R.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Stratton, Kelly G.; Cockrell, Adam S.; Debbink, Kari; Sims, Amy C.; Waters, Katrina M.; Baric, Ralph S.; Fernandez-Sesma, Ana

    2017-11-15

    ABSTRACT

    Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.

    IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that

  18. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    Science.gov (United States)

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus.

    Science.gov (United States)

    Wu, Xiaodong; Qi, Xian; Liang, Mifang; Li, Chuan; Cardona, Carol J; Li, Dexin; Xing, Zheng

    2014-06-01

    Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNβ-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses. © FASEB.

  20. Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

    Directory of Open Access Journals (Sweden)

    Chiu-Ping Fang

    Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

  1. Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase.

    Science.gov (United States)

    Ingravallo, P; Lahser, F; Xia, E; Sodowich, B; Lai, V C; Hong, Z; Zhong, W

    2001-06-01

    The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

  2. Emergence of a virulent porcine reproductive and respiratory syndrome virus (PRRSV 1 strain in Lower Austria

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    Leonie J Sinn

    2016-11-01

    Full Text Available Abstract Background In spring 2015, an outbreak of porcine reproductive and respiratory syndrome (PRRS struck Lower Austria caused by a PRRS virus (PRRSV strain spreading rapidly among both previously PRRSV negative and vaccinated pig herds. This case report describes the first well-documented emergence of the PRRSV strain responsible for this outbreak. Case presentation A PRRSV seronegative piglet-producing farm in Lower Austria encountered losses in foetuses and suckling piglets of up to 90 %; clinical signs in sows and nursery piglets included fever and reduced feed intake. Additionally, high percentages of repeat breeders and losses of up to 40 % in nursery piglets occurred. An infection with PRRSV was suggested by the detection of antibodies by enzyme linked immunosorbent assay and confirmed by quantitative real time PCR. The underlying PRRSV strain, termed AUT15-33, was isolated by passage on porcine alveolar macrophages, partially sequenced (ORF2-7 and grouped as PRRSV-1, subtype 1. In phylogenetic analysis of the genome region coding for the structural proteins, ORF2-7, AUT15-33 clustered with Belgian strains but identities were as low as 88 %. In contrast, analysis of ORF7 sequences revealed a close relationship to Croatian strains from 2012 with an identity of 94 – 95 %. Conclusions In the year following the outbreak, the same PRRSV strain was identified repeatedly in different regions of Austria. It can be speculated that the new strain has novel advantageous properties.

  3. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

    Science.gov (United States)

    Ikegami, Tetsuro; Peters, C J; Makino, Shinji

    2005-05-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

  4. The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

    Directory of Open Access Journals (Sweden)

    Laura Martinez-Alvarez

    2013-06-01

    Full Text Available A hallmark of group/species A rotavirus (RVA replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1 is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV. NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

  5. Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Brocchi, E.; Bergmann, I.E.; Dekker, A.

    2006-01-01

    To validate the use of serology in substantiating freedom from infection after foot-and-mouth disease (FMD) outbreaks have been controlled by measures that include vaccination, 3551 sera were tested with six assays that detect antibodies to the non-structural proteins of FMD virus. The sera came...

  6. Antigenicity of envelop and non-structural proteins of dengue serotypes and their potentiality to elicit specifi antibody

    Directory of Open Access Journals (Sweden)

    Ramesh Venkatachalam

    2015-06-01

    Full Text Available Objective: To find out the antigenic nature of envelop (E and non-structural (NS proteins and their ability to induce specific antibodies, and to investigate specific antibody produced by specific dengue virus (DENV serotypes. Methods: Amino acid sequences of E and NS proteins of dengue serotypes were analysed by using VaxiJen antigen predicition server. The transmembrane of topology analyses were conducted by using transmembrane prediction using hidden markov models. The Hex dock server was used for docking. Results: The antigenicity score and exomembrane potentiality of E and NS proteins were calculated. All those proteins were antigenic; these antigens were made to interact with antibodies such as immunoglobulin A, immunoglobulin G and immunoglobulin M. Higher energy values of immunoglobulin M were found in DENV-1 and DENV-2, and more energy values were found in immunoglobulin G of DENV-3, DENV-4, NS-1, NS-3 and NS-5. Conclusions: In the present study, DENV-1 and DENV-2 are positive to immunoglobulin M and involved in the primary infection. DENV 3, DENV 4 and all the NS proteins (NS-1, NS-3, NS-5 which elicit immunoglobulin G are involved in the secondary infection.

  7. The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.

    Science.gov (United States)

    Tang, Fenfen; Xia, Hongjie; Wang, Peipei; Yang, Jie; Zhao, Tianyong; Zhang, Qi; Hu, Yuanyang; Zhou, Xi

    2014-09-01

    Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Respiratory Syncytial Virus Nonstructural Proteins Upregulate SOCS1 and SOCS3 in the Different Manner from Endogenous IFN Signaling

    Directory of Open Access Journals (Sweden)

    Junwen Zheng

    2015-01-01

    Full Text Available Respiratory syncytial virus (RSV infection upregulates genes of the suppressor of cytokine signaling (SOCS family, which utilize a feedback loop to inhibit type I interferon dependent antiviral signaling pathway. Here, we reconstituted RSV nonstructural (NS protein expression plasmids (pNS1, pNS2, and pNS1/2 and tested whether NS1 or NS2 would trigger SOCS1 and SOCS3 protein expression. These NS proteins inhibited interferon- (IFN- α signaling through a mechanism involving the induction of SOCS1 and SOCS3, which appeared to be different from autocrine IFN dependent. NS1 induced both SOCS1 and SOCS3 upregulation, while NS2 only induced SOCS1 expression. The induced expression of SOCS1 and SOCS3 preceded endogenous IFN-signaling activation and inhibited the IFN-inducible antiviral response as well as chemokine induction. Treatments with INF-α and NS proteins both induced SOCS1 expression; however, they had opposing effects on IFN-α-dependent antiviral gene expression. Our results indicate that NS1 and NS2, which induce the expression of SOCS1 or SOCS3, might represent an independent pathway of stimulating endogenous IFN signaling.

  9. Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2010-08-01

    Full Text Available In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV, is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20 and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20 were blocked in encapsidation (no virus after blind passages but could be rescued if the capsid and 2C(ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i genome replication is known to be stringently linked to translation, (ii morphogenesis is known to be stringently linked to genome replication, (iii newly synthesized 2C(ATPase is an essential component of the replication complex, and (iv 2C(ATPase has specific affinity to capsid protein(s. These conditions lead to morphogenesis at the site where newly

  10. T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

    Science.gov (United States)

    Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

    2017-12-01

    The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

  11. Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.

    Science.gov (United States)

    Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

    2017-03-01

    Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

  12. Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

    Science.gov (United States)

    Li, Dan; Lei, Caoqi; Xu, Zhisheng; Yang, Fan; Liu, Huanan; Zhu, Zixiang; Li, Shu; Liu, Xiangtao; Shu, Hongbing; Zheng, Haixue

    2016-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system. PMID:26883855

  13. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Science.gov (United States)

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  14. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Directory of Open Access Journals (Sweden)

    Satoshi Sekiguchi

    Full Text Available Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV, is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis, liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25, which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-/MxCre((+/- mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor TNF-α and (interleukin IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  15. Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

    Science.gov (United States)

    Khasnatinov, Maxim A; Tuplin, Andrew; Gritsun, Dmitri J; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Labuda, Milan; Gould, Ernest A; Gritsun, Tamara S

    2016-01-01

    Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

  16. Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Maxim A Khasnatinov

    Full Text Available Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV. The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS, Vs and Hypr induce low and high cytopathic effects (cpe, respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

  17. Flaviviridae virus nonstructural proteins 5 and 5A mediate viral immune evasion and are promising targets in drug development.

    Science.gov (United States)

    Chen, Shun; Yang, Chao; Zhang, Wei; Mahalingam, Suresh; Wang, Mingshu; Cheng, Anchun

    2018-05-06

    Infections with viruses in the Flaviviridae family have a vast global and economic impact because of the high morbidity and mortality. The pathogenesis of Flaviviridae infections is very complex and not fully understood because these viruses can inhibit multiple immune pathways including the complement system, NK cells, and IFN induction and signalling pathways. The non-structural (NS) 5 and 5A proteins of Flaviviridae viruses are highly conserved and play an important role in resisting host immunity through various evasion mechanisms. This review summarizes the strategies used by the NS5 and 5A proteins of Flaviviridae viruses for evading the innate immune response by inhibiting pattern recognition receptor (PRR) signalling pathways (TLR/MyD88, IRF7), suppressing interferon (IFN) signalling pathways (IFN-γRs, STAT1, STAT2), and impairing the function of IFN-stimulated genes (ISGs) (e.g. protein kinase R [PKR], oligoadenylate synthase [OAS]). All of these immune evasion mechanisms depend on the interaction of NS5 or NS5A with cellular proteins, such as MyD88 and IRF7, IFN-αRs, IFN-γRs, STAT1, STAT2, PKR and OAS. NS5 is the most attractive target for the discovery of broad spectrum compounds against Flaviviridae virus infection. The methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities of NS5 are the main therapeutic targets for antiviral drugs against Flaviviridae virus infection. Based on our site mapping, the sites involved in immune evasion provide some potential and promising targets for further novel antiviral therapeutics. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Development of an indirect ELISA with epitope on nonstructural protein of Muscovy duck parvovirus for differentiating between infected and vaccinated Muscovy ducks.

    Science.gov (United States)

    Yan, B; Ma, J-Z; Yu, T-F; Shao, S-L; Li, M; Fan, X-D

    2014-12-01

    The aim of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of Muscovy duck parvovirus (MDPV). Sera (100) from negative and vaccinated Muscovy ducks were compared with infected sera (240) to establish the cut-off value of this i-ELISA. There was a significant difference between the positive and negative populations (P ducks from Muscovy ducks vaccinated with inactivated virus. In this study, we developed an i-ELISA based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of MDPV. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. © 2014 The Society for Applied Microbiology.

  19. Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

    Directory of Open Access Journals (Sweden)

    Da Ao

    Full Text Available Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER, with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

  20. Nuclear import inhibitor N-(4-hydroxyphenyl) retinamide targets Zika virus (ZIKV) nonstructural protein 5 to inhibit ZIKV infection.

    Science.gov (United States)

    Wang, Chunxiao; Yang, Sundy N Y; Smith, Kate; Forwood, Jade K; Jans, David A

    2017-12-02

    In the absence of approved therapeutics, Zika virus (ZIKV)'s recent prolific outbreaks in the Americas, together with impacts on unborn fetuses of infected mothers, make it a pressing human health concern worldwide. Although a key player in viral replication in the infected host cell cytoplasm, ZIKV non-structural protein 5 (NS5) appears to contribute integrally to pathogenesis by localising in the host cell nucleus, in similar fashion to NS5 from Dengue virus (DENV). We show here for the first time that ZIKV NS5 is recognized with high nanomolar affinity by the host cell importin α/β1 heterodimer, and that this interaction can be blocked by the novel DENV NS5 targeting inhibitor N-(4-hydroxyphenyl) retinamide (4-HPR). Importantly, we show that 4-HPR has potent anti-ZIKV activity at low μM concentrations. With an established safety profile for human use, 4-HPR represents an exciting possibility as an anti-ZIKV agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

    Science.gov (United States)

    Bhushan, Lokesh; Abraham, Ambily; Choudhury, Nirupam Roy; Rana, Vipin Singh; Mukherjee, Sunil Kumar; Savithri, Handanahal Subbarao

    2015-04-01

    The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

  2. The nonstructural protein NSs induces a variable antibody response in domestic ruminants naturally infected with Rift Valley fever virus.

    Science.gov (United States)

    Fernandez, José-Carlos; Billecocq, Agnès; Durand, Jean Paul; Cêtre-Sossah, Catherine; Cardinale, Eric; Marianneau, Philippe; Pépin, Michel; Tordo, Noël; Bouloy, Michèle

    2012-01-01

    Rift Valley fever (RVF) is an emerging zoonosis in Africa which has spread to Egypt, the Arabian Peninsula, Madagascar, and Comoros. RVF virus (RVFV) (Bunyaviridae family, Phlebovirus genus) causes a wide range of symptoms in humans, from benign fever to fatal hemorrhagic fever. Ruminants are severely affected by the disease, which leads to a high rate of mortality in young animals and to abortions and teratogenesis in pregnant females. Diagnostic tests include virus isolation and genome or antibody detection. During RVFV infection, the nucleoprotein encapsidating the tripartite RNA genome is expressed in large amounts and raises a robust antibody response, while the envelope glycoproteins elicit neutralizing antibodies which play a major role in protection. Much less is known about the antigenicity/immunogenicity of the nonstructural protein NSs, which is a major virulence factor. Here we have developed a competitive enzyme-linked immunosorbent assay (ELISA) enabling detection of low levels of NSs-specific antibodies in naturally infected or vaccinated ruminants. Detection of the NSs antibodies was validated by Western blotting. Altogether, our data showed that the NSs antibodies were detected in only 55% of animals naturally infected by RVFV, indicating that NSs does not induce a consistently high immune response. These results are discussed in light of differentiation between infected and vaccinated animals (DIVA) tests distinguishing naturally infected animals and those vaccinated with NSs-defective vaccines.

  3. Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus.

    Science.gov (United States)

    Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Mir, Carmen; Gebe, John A; Admon, Arie; López, Daniel

    2016-06-01

    Proper antiviral humoral and cellular immune responses require previous recognition of viral antigenic peptides that are bound to HLA class II molecules, which are exposed on the surface of antigen-presenting cells. The helper immune response is critical for the control and the clearance of human respiratory syncytial virus (HRSV) infection, a virus with severe health risk in infected pediatric, immunocompromised, and elderly populations. In this study, using a mass spectrometry analysis of complex HLA class II-bound peptide pools that were isolated from large amounts of HRSV-infected cells, 19 naturally processed HLA-DR ligands, most of them included in a complex nested set of peptides, were identified. Both the immunoprevalence and the immunodominance of the HLA class II response to HRSV were focused on one nonstructural (NS1) and two structural (matrix and mainly fusion) proteins of the infective virus. These findings have clear implications for analysis of the helper immune response as well as for antiviral vaccine design. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India

    Directory of Open Access Journals (Sweden)

    Raghavendra Sumanth Pudupakam

    2017-03-01

    Full Text Available Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3 gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV. This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2 to elucidate its genetic relationship to global BTV isolates. Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

  5. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Science.gov (United States)

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  6. The nonstructural proteins of Nipah virus play a key role in pathogenicity in experimentally infected animals.

    Directory of Open Access Journals (Sweden)

    Misako Yoneda

    Full Text Available Nipah virus (NiV P gene encodes P protein and three accessory proteins (V, C and W. It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V-, rNiV(C-, and rNiV(W-, respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V- and rNiV(C- were lower than the other recombinants. The rNiV(V-, rNiV(C- and rNiV(W- suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V- and rNiV(C- but not the rNiV(W- virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.

  7. Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus.

    Science.gov (United States)

    Goswami, Suneha; Sahana, Nandita; Pandey, Vanita; Doblas, Paula; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

    2012-01-01

    Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis*

    Science.gov (United States)

    Barnwal, Bhaskar; Karlberg, Helen; Mirazimi, Ali; Tan, Yee-Joo

    2016-01-01

    Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells. PMID:26574543

  9. Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

    Science.gov (United States)

    Sun, Qiyu; Qi, Xian; Zhang, Yan; Wu, Xiaodong; Liang, Mifang; Li, Chuan; Li, Dexin; Cardona, Carol J; Xing, Zheng

    2016-07-29

    Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs*

    Science.gov (United States)

    Sun, Qiyu; Qi, Xian; Zhang, Yan; Wu, Xiaodong; Liang, Mifang; Li, Chuan; Li, Dexin; Cardona, Carol J.; Xing, Zheng

    2016-01-01

    Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. PMID:27226560

  11. The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses

    DEFF Research Database (Denmark)

    Mokhtar, Helen; Pedrera, Miriam; Frossard, Jean-Pierre

    2016-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses...... proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered...... attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development....

  12. Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells

    International Nuclear Information System (INIS)

    Garcia-Briones, Mercedes; Rosas, Maria F.; Gonzalez-Magaldi, Monica; Martin-Acebes, Miguel A.; Sobrino, Francisco; Armas-Portela, Rosario

    2006-01-01

    Differences in the kinetics of expression and cell distribution among FMDV non-structural proteins (NSPs) have been observed in BHK-21-infected cells. 3D pol was the first protein detected by immunofluorescence (1.5 h p.i.), showing a perinuclear distribution. At 2-2.5 h p.i., 2B, 2C, 3B and 3C were detected, mostly exhibiting a punctuated, scattered pattern, while 3A and 3D pol appeared concentrated at one side of the nucleus. This distribution was exhibited by all the NSPs from 3 h p.i., being 2C and, to a lesser extent, precursors 2BC and 3ABBB, the only proteins detected by Western blotting at that infection time. From 4 h p.i., all mature NSPs as well as precursors 2BC, 3ABBB, 3ABB, 3AB and 3CD pol were detected by this technique. In spite of their similar immunofluorescence patterns, 2C and 3A co-localized partially by confocal microscopy at 3.5 h p.i., and 3A, but not 2C, co-localized with the ER marker calreticulin, suggesting differences in the distribution of these proteins and/or their precursors as infection proceeded. Transient expression of 2C and 3AB resulted in punctuated fluorescence patterns similar to those found in early infected cells, while 3A showed a more diffuse distribution. A shift towards a fibrous pattern was noticed for 3ABB, while a major change was observed in cells expressing 3ABBB, which displayed a perinuclear fibrous distribution. Interestingly, when co-expressed with 3D pol , the pattern observed for 3ABBB fluorescence was altered, resembling that exhibited by cells transfected with 3AB. Transient expression of 3D pol showed a homogeneous cell distribution that included, as determined by confocal microscopy, the nucleus. This was confirmed by the detection of 3D pol in nuclear fractions of transfected cells. 3D pol and its precursor 3CD pol were also detected in nuclear fractions of infected cells, suggesting that these proteins can directly interact with the nucleus during FMDV infection

  13. Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs.

    Science.gov (United States)

    Tilston-Lunel, Natasha L; Acrani, Gustavo Olszanski; Randall, Richard E; Elliott, Richard M

    2015-12-23

    Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet

  14. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    Science.gov (United States)

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  15. Raman molecular fingerprint of non-structural protein 1 in phosphate buffer saline with gold substrate.

    Science.gov (United States)

    Radzol, A R M; Lee, Khuan Y; Mansor, W

    2013-01-01

    SERS is a form of Raman spectroscopy that is enhanced with nano-sensing chip as substrate. It can yield distinct biochemical fingerprint for molecule of solids, liquids and gases. Vice versa, it can be used to identify unknown molecule. It has further advantage of being non-invasive, non-contact and cheap, as compared to other existing laboratory based techniques. NS1 has been clinically accepted as an alternative biomarker to IgM in diagnosing viral diseases carried by virus of flaviviridae. Its presence in the blood serum at febrile stage of the flavivirus infection has been proven. Being an antigen, it allows early detection that can help to reduce the mortality rate. This paper proposes SERS as a technique for detection of NS1 from its scattering spectrum. Contribution from our work so far has never been reported. From our experiments, it is found that NS1 protein is Raman active. Its spectrum exhibits five prominent peaks at Raman shift of 548, 1012, 1180, 1540 and 1650 cm(-1). Of these, peak at 1012 cm(-1) scales the highest intensity. It is singled out as the peak to fingerprint the NS1 protein. This is because its presence is verified by the ring breathing vibration of the benzene ring structure side chain molecule. The characteristic peak is found to vary in proportion to concentration. It is found that for a 99% change in concentration, a 96.7% change in intensity is incurred. This yields a high sensitivity of about one a.u. per ppm. Further investigation from the characterization graph shows a correlation coefficient of 0.9978 and a standard error estimation of 0.02782, which strongly suggests a linear relationship between the concentration and characteristic peak intensity of NS1. Our finding produces favorable evidence to the use of SERS technique for detection of NS1 protein for early detection of flavivirus infected diseases with gold substrate.

  16. Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

    Directory of Open Access Journals (Sweden)

    Encheng Sun

    Full Text Available West Nile virus (WNV is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1 of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24 were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV, Newcastle Disease Virus (NDV, Duck Plague Virus (DPV and Goose Parvovirus (GPV antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and

  17. La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts.

    Science.gov (United States)

    Blakqori, Gjon; Delhaye, Sophie; Habjan, Matthias; Blair, Carol D; Sánchez-Vargas, Irma; Olson, Ken E; Attarzadeh-Yazdi, Ghassem; Fragkoudis, Rennos; Kohl, Alain; Kalinke, Ulrich; Weiss, Siegfried; Michiels, Thomas; Staeheli, Peter; Weber, Friedemann

    2007-05-01

    La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

  18. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    Science.gov (United States)

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  19. Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

    Energy Technology Data Exchange (ETDEWEB)

    Karttunen, Jenni; Mäntynen, Sari [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Ihalainen, Teemu O. [Stem Cells in Neurological Applications Group, BioMediTech, University of Tampere, Tampere (Finland); Bamford, Jaana K.H. [Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, P.O. Box 35, 40014 Jyväskylä (Finland); Oksanen, Hanna M., E-mail: hanna.oksanen@helsinki.fi [Institute of Biotechnology and Department of Biosciences, University of Helsinki, Biocenter 2, P.O. Box 56 (Viikinkaari 5), FIN-00014 Helsinki (Finland)

    2015-08-15

    Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.

  20. Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

    Science.gov (United States)

    Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

    2016-06-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of

  1. Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

    Directory of Open Access Journals (Sweden)

    Chao-Kuen Lai

    Full Text Available Nonstructural protein 5A (NS5A of hepatitis C virus (HCV serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

  2. Engineering a CTL-Tailored Replicon RNA Vaccine against PRRSV

    DEFF Research Database (Denmark)

    Welner, Simon; Werder, Simea; Nielsen, Morten

    The development of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) has been hampered by the high mutation rate and the multiple immunoevasive strategies of the virus. With the overall aim of designing a broad coverage vaccine that induces an effective CTL response aga...... will be available for IVIS. This study exemplifies how bioinformatics epitope prediction, recombinant SLA molecules and RNA virus replicon design can be used to engineer a replicating non-propagating vaccine tailored to deliver conserved and immunogenic CTL epitopes....... against PRRSV, we have used a bioinformatics approach to identify common PRRSV type 2 epitopes predicted to react broadly with predominant swine MHC (SLA) alleles. All possible 9- and 10-mer peptides derived from 104 wild-type strains were analyzed in silico for their predicted binding affinity to 3...... cloned into a classical swine fever virus (CSFV)-derived replicon vector. Virus replicon particles (VRP) were rescued by transfection of a complementing cell line with replicon RNA. Polyepitope expression and subsequent proteasomal degradation was confirmed indirectly by increased FLAG-tagged protein...

  3. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Fogeron, Marie-Laure [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Jirasko, Vlastimil; Penzel, Susanne [ETH Zurich, Physical Chemistry (Switzerland); Paul, David [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Montserret, Roland; Danis, Clément; Lacabanne, Denis; Badillo, Aurélie [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Gouttenoire, Jérôme; Moradpour, Darius [University of Lausanne, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois (Switzerland); Bartenschlager, Ralf [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Penin, François [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); and others

    2016-06-15

    We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [{sup 2}H,{sup 13}C,{sup 15}N]-labeled protein are shown to yield narrow {sup 13}C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

  4. Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

    International Nuclear Information System (INIS)

    Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

    2010-01-01

    The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1α and nsp1β subunits. In infected cells, we detected the actual existence of nsp1α and nsp1β. Cleavage sites between nsp1α/nsp1β and nsp1β/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1α and nsp1β mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-β expression. The nsp1β was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1β has ability to inhibit both interferon synthesis and signaling, while nsp1α alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

  5. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  6. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    International Nuclear Information System (INIS)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-01-01

    Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  7. Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

    Science.gov (United States)

    Zhou, Jing; Chen, Jing; Zhang, Xiao-Min; Gao, Zhi-Can; Liu, Chun-Chun; Zhang, Yun-Na; Hou, Jin-Xiu; Li, Zhao-Yao; Kan, Lin; Li, Wen-Liang; Zhou, Bin

    2018-04-01

    Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S -transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B. IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo , but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities. Copyright © 2018 American Society for Microbiology.

  8. Phylogenetic Analysis of PRRSV from Danish Pigs

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard; Larsen, Lars Erik

    , named from their geographic origin of identification. Great diversity within the two genotypes exists, and further division of PRRSV EU type into at least 3 subtypes has been suggested (Stadejek et al. 2006, 2008). In Denmark PRRSV EU type was first identified in 1992 and a few years later the US type......-PCR, essentially as described by Egli et al. 2001, on RNA extracted with RNeasy Mini Kit (QIAGEN). Complete open reading frames (ORF) ORF5 and ORF7 were PCR amplified as described (Oleksiewicz et al. 1998) and sequenced. Sequences were aligned and Neighbour-Joining trees were constructed with ClustalX. Trees were...

  9. Interaction Research on the Antiviral Molecule Dufulin Targeting on Southern Rice Black Streaked Dwarf Virus P9-1 Nonstructural Protein

    Directory of Open Access Journals (Sweden)

    Zhenchao Wang

    2015-03-01

    Full Text Available ern rice black streaked dwarf virus (SRBSDV causes severe harm to rice production. Unfortunately, studies on effective antiviral drugs against SRBSDV and interaction mechanism of antiviral molecule targeting on SRBSDV have not been reported. This study found dufulin (DFL, an ideal anti-SRBSDV molecule, and investigated the interactions of DFL targeting on the nonstructural protein P9-1. The biological sequence information and bonding characterization of DFL to four kinds of P9-1 protein were described with fluorescence titration (FT and microscale thermophoresis (MST assays. The sequence analysis indicated that P9-1 had highly-conserved C- and N-terminal amino acid residues and a hypervariable region that differed from 131 aa to 160 aa. Consequently, wild-type (WT-His-P9-1, 23 C-terminal residues truncated (TR-ΔC23-His-P9-1, 6 N-terminal residues truncated (TR-ΔN6-His-P9-1, and Ser138 site-directed (MU-138-His-P9-1 mutant proteins were expressed. The FT and MST assay results indicated that DFL bounded to WT-His-P9-1 with micromole affinity and the 23 C-terminal amino acids were the potential targeting site. This system, which combines a complete sequence analysis, mutant protein expression, and binding action evaluating system, could further advance the understanding of the interaction abilities between antiviral drugs and their targets.

  10. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine

    This PhD thesis presents the diversity of Porcine Reproductive and Respiratory Syndrome viruses (PRRSV) circulating in the Danish pig population. PRRS is a disease in pigs caused by the PRRS virus resulting in reproductive failures in sows and gilts and respiratory diseases in pigs . Due to genetic...

  11. Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

    Science.gov (United States)

    Rezelj, Veronica V; Överby, Anna K; Elliott, Richard M

    2015-05-01

    Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a

  12. Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein.

    Science.gov (United States)

    Wang, X X; Wang, F X; Li, Z G; Wen, Y J; Wang, X; Song, N; Wu, H

    2018-01-01

    An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41-17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92. Copyright © 2017. Published by Elsevier B.V.

  13. In silico targeting of non-structural 4B protein from dengue virus 4 with spiropyrazolopyridone: study of molecular dynamics simulation, ADMET and virtual screening.

    Science.gov (United States)

    Hussain, Waqar; Qaddir, Iqra; Mahmood, Sajid; Rasool, Nouman

    2018-06-01

    Dengue fever is one of the most prevalent disease in tropical and sub-tropical regions of the world. According to the World Health Organisation (WHO), approximately 3.5 billion people have been affected with dengue fever. Four serotypes of dengue virus (DENV) i.e. DENV1, DENV2, DENV3 and DENV4 have up to 65% genetic variations among themselves. dengue virus 4 (DENV4) was first reported from Amazonas, Brazil and is spreading perilously due to lack of awareness of preventive measures, as it is the least targeted serotype. In this study, non-structural protein 4B of dengue virus 4 (DENV4-NS4B) is computationally characterised and simulations are performed including solvation, energy minimizations and neutralisation for the refinement of predicted model of the protein. The spiropyrazolopyridone is considered as an effective drug against NS4B of DENV2, therefore, a total of 91 different analogues of spiropyrazolopyridone are used to analyse their inhibitory action against DENV4-NS4B. These compounds are docked at the binding site with various binding affinities, representing their efficacy to block the binding pocket of the protein. Pharmacological and pharmacokinetic assessment performed on these inhibitors shows that these are suitable candidates to be used as a drug against the dengue fever. Among all these 91 compounds, Analogue-I and Analogue-II are analysed to be the most effective inhibitor having potential to be used as drugs against dengue virus.

  14. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  15. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    International Nuclear Information System (INIS)

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-01-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus

  16. Seismic analysis of nonstructural elements

    OpenAIRE

    Toledo Arias, Carlos Alberto

    2013-01-01

    Nonstructural failures have accounted for the majority of earthquake damage in several recent earthquakes. Thus, it is critical to raise awareness of potential nonstructural risks, the costly consequences of nonstructural failures, and the opportunities that exist to limit future losses. Non-structural parts of a building have the potential to modify earthquake response of the primary structure in an unplanned way. This can lead to severe structural damage or even collapse. Failure of non-str...

  17. Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome.

    Science.gov (United States)

    Parvaiz, Fahed; Manzoor, Sobia; Iqbal, Jawed; McRae, Steven; Javed, Farrakh; Ahmed, Qazi Laeeque; Waris, Gulam

    2014-05-01

    Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

  18. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    Energy Technology Data Exchange (ETDEWEB)

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina); Vas, Mariana del, E-mail: mdelvas@cnia.inta.gov.ar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina)

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  19. Nonstructural NSs protein of rift valley fever virus interacts with pericentromeric DNA sequences of the host cell, inducing chromosome cohesion and segregation defects.

    Science.gov (United States)

    Mansuroglu, Z; Josse, T; Gilleron, J; Billecocq, A; Leger, P; Bouloy, M; Bonnefoy, E

    2010-01-01

    Rift Valley fever virus (RVFV) is an emerging, highly pathogenic virus; RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemorrhagic fever in humans, as well as death, abortions, and fetal deformities in animals. RVFV nonstructural NSs protein, a major factor of the virulence, forms filamentous structures in the nuclei of infected cells. In order to further understand RVFV pathology, we investigated, by chromatin immunoprecipitation, immunofluorescence, fluorescence in situ hybridization, and confocal microscopy, the capacity of NSs to interact with the host genome. Our results demonstrate that even though cellular DNA is predominantly excluded from NSs filaments, NSs interacts with some specific DNA regions of the host genome such as clusters of pericentromeric gamma-satellite sequence. Targeting of these sequences by NSs was correlated with the induction of chromosome cohesion and segregation defects in RVFV-infected murine, as well as sheep cells. Using recombinant nonpathogenic virus rZHDeltaNSs210-230, expressing a NSs protein deleted of its region of interaction with cellular factor SAP30, we showed that the NSs-SAP30 interaction was essential for NSs to target pericentromeric sequences, as well as for induction of chromosome segregation defects. The effect of RVFV upon the inheritance of genetic information is discussed with respect to the pathology associated with fetal deformities and abortions, highlighting the main role played by cellular cofactor SAP30 on the establishment of NSs interactions with host DNA sequences and RVFV pathogenesis.

  20. Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

    Science.gov (United States)

    Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

    2011-01-01

    Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

  1. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    Science.gov (United States)

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  2. Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

    DEFF Research Database (Denmark)

    Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih

    2002-01-01

    Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased....

  3. Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver.

    Directory of Open Access Journals (Sweden)

    Takeya Tsutsumi

    Full Text Available The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2'-5' oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity.

  4. Chemiluminescence Immunoassay for the Detection of Antibodies against the 2C and 3ABC Nonstructural Proteins Induced by Infecting Pigs with Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Liu, Zezhong; Shao, Junjun; Zhao, Furong; Zhou, Guangqing; Gao, Shandian; Liu, Wei; Lv, Jianliang; Li, Xiumei; Li, Yangfan; Chang, Huiyun; Zhang, Yongguang

    2017-08-01

    The potential diagnostic value of chemiluminescence immunoassays (CLIAs) has been accepted in recent years, although their use for foot-and-mouth disease (FMD) diagnostics has not been reported. Full-length 3ABC and 2C proteins were expressed in bacteria and purified by affinity chromatography to develop a rapid and accurate approach to distinguish pigs infected with foot-and-mouth disease virus (FMDV) from vaccinated pigs. The recombinant proteins were then used as antigens to develop two CLIAs for the detection of antibodies against nonstructural viral proteins. The diagnostic performance of the two assays was compared by analyzing serum from pigs (naive pigs, n = 63; vaccinated, uninfected pigs, n = 532; naive, infected pigs, n = 117) with a known infection status. The 3ABC-2C CLIA had a higher accuracy rate, with a diagnostic sensitivity of 100% and a diagnostic specificity of 96.5%, than the 3ABC CLIA, which had a diagnostic sensitivity of 95.7% and a diagnostic specificity of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected infection in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections. Copyright © 2017 American Society for Microbiology.

  5. Comparative study and grouping of nonstructural (NS1)proteins of influenza A viruses by the method of oligopeptide mapping

    International Nuclear Information System (INIS)

    Sokolov, B.P.; Rudneva, I.A.; Zhdanov, V.M.

    1983-01-01

    Oligopeptide mapping of 35 S-methionine labeled non-stuctural (NS1) proteins of 23 influenza A virus strains showed the presence of both common and variable oligopeptides. Analysis of the oligopeptide maps revealed at least four groups of NS1 proteins. The first group includes NS1 proteins of several human H1N1 influenza viruses (that were designated as H0N1 according to the old classification). The second group is composed of NS1 proteins of H1N1 and H2N2 viruses. The third group includes NS1 proteins of H3N2 human influenza viruses. The fourth group is composed of NS1 proteins of five avian influenza viruses and an equine (H3N8) influenza virus. Two animal influenza viruses A/equi/Prague/56 (H7N7) and A/duck/England/56 (H11N6) contain NS1 proteins that belong to the second group. (Author)

  6. Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs.

    Directory of Open Access Journals (Sweden)

    Masayuki Sano

    Full Text Available Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp. Because of the capacity of Sendai virus (SeV nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.

  7. Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc...

  8. Production of Polyclonal Antiobies to a Recombinant Potato Mop-top Virus Non-structural Triple Gene Block Protein l

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Filigarová, Marie; Pečenková, Tamara

    2006-01-01

    Roč. 154, - (2006), s. 422-427 ISSN 0931-1785 R&D Projects: GA ČR GA522/04/1329 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato mop-top virus * recombinant protein * triple gene block * polyclonal antibodies Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.817, year: 2006

  9. Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

    Science.gov (United States)

    Tran, Si C.; Pham, Tu M.; Nguyen, Lam N.; Park, Eun-Mee; Lim, Yun-Sook

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of

  10. The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion.

    Science.gov (United States)

    Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling; Liao, Ching-Len

    2016-02-01

    NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained

  11. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    International Nuclear Information System (INIS)

    Sun, Bin; Cai, Yingyue; Li, Yongshu; Li, Jingjing; Liu, Kaiyu; Li, Yi; Yang, Yongbo

    2013-01-01

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization

  12. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  13. Efficacy of type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs

    Science.gov (United States)

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant reproductive losses in the sow herd and respiratory disease in growing pigs. It is a virus that belongs to the family Arteriviridae virus for which there are two major genotypes, Type 1 represented by Lelystad virus, the ...

  14. Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

    Science.gov (United States)

    Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

    2016-09-15

    A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection

  15. Effects of nonstructural carbohydrates and protein sources on intake, apparent total tract digestibility, and ruminal metabolism in vivo and in vitro with high-concentrate beef cattle diets.

    Science.gov (United States)

    Rotger, A; Ferret, A; Calsamiglia, S; Manteca, X

    2006-05-01

    To investigate the effects of synchronizing nonstructural carbohydrate (NSC) and protein degradation on intake and rumen microbial fermentation, four ruminally fistulated Holstein heifers (BW = 132.3 +/- 1.61 kg) fed high-concentrate diets were assigned to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments studied in vivo and in vitro with a dual-flow continuous culture system. Two NSC sources (barley and corn) and 2 protein sources [soybean meal (SBM) and sunflower meal (SFM)] differing in their rate and extent of ruminal degradation were combined resulting in a synchronized rapid fermentation diet (barley-SFM), a synchronized slow fermentation diet (corn-SBM), and 2 unsynchronized diets with a rapidly and a slowly fermenting component (barley-SBM, and corn-SFM). In vitro, the fermentation profile was studied at a constant pH of 6.2, and at a variable pH with 12 h at pH 6.4 and 12 h at pH 5.8. Synchronization tended to result in greater true OM digestion (P = 0.072), VFA concentration (P = 0.067), and microbial N flow (P = 0.092) in vitro, but had no effects on in vivo fermentation pattern or on apparent total tract digestibility. The NSC source affected the efficiency of microbial protein synthesis in vitro, tending to be greater (P = 0.07) for barley-based diets, and in vivo, the NSC source tended to affect intake. Dry matter and OM intake tended to be greater (P > or = 0.06) for corn- than barley-based diets. Ammonia N concentration was lower in vitro (P = 0.006) and tended to be lower in vivo (P = 0.07) for corn- than barley-based diets. In vitro, pH could be reduced from 6.4 to 5.8 for 12 h/d without any effect on ruminal fermentation or microbial protein synthesis. In summary, ruminal synchronization seemed to have positive effects on in vitro fermentation, but in vivo recycling of endogenous N or intake differences could compensate for these effects.

  16. Pegylated interferon and ribavirin promote early evolution of nonstructural 5A protein in individuals with hepatitis C who demonstrate a response to treatment.

    Science.gov (United States)

    Jain, Mamta K; Yuan, He-Jun; Adams-Huet, Beverley; Reeck, Amanda; Shelton, Janel; Attar, Nahid; Zhang, Song; Neumann, Avidan U; Carney, David S; Gale, Michael; Lee, William M

    2009-09-15

    Hepatitis C virus (HCV) quasispecies diversity is more likely to affect early viral decline during treatment of hepatitis C than is having human immunodeficiency virus (HIV) infection. We evaluated the influence of HCV therapy on changes in the nonstructural 5A (NS5A) protein. Fifteen patients with HCV genotype 1 infection with or without HIV infection were recruited for the present study, and the decrease in the HCV RNA level was measured at early time points. The evolution of HCV NS5A quasispecies within the first week was analyzed by comparing the clones observed at later times in the study with the baseline consensus sequence of individual patients. The response to therapy was defined as an early response (ER; ie, an HCV RNA level <615 IU/mL at week 4) or a slow response (SR; ie, a detectable HCV RNA level at week 4). HIV infection did not affect early viral kinetics. At baseline, lower diversity was seen in NS5A and in the amino and carboxyl termini of patients with an ER, compared with those with an SR. Rapid evolution of the NS5A genetic region occurred in patients with an ER (P = .01) but not in those with an SR (P = .73). The evolution was the result of an increase in the number of amino acid substitutions in the carboxyl region (P = .02) in patients with an ER. Selective pressure appears to result in more-marked changes in individuals with an ER than in those with an SR. The carboxyl terminus was subject to the most change and may be an important determinant of phenotypic resistance to interferon-based therapy.

  17. Modification of S-Adenosyl-l-Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation.

    Science.gov (United States)

    Tambunan, Usman Sumo Friend; Nasution, Mochammad Arfin Fardiansyah; Azhima, Fauziah; Parikesit, Arli Aditya; Toepak, Erwin Prasetya; Idrus, Syarifuddin; Kerami, Djati

    2017-01-01

    Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world's population in tropical and subtropical countries. Nonstructural protein 5 (NS5) methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S -adenosyl-l-methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2'OH, resulting in S -adenosyl-l-homocysteine (SAH). The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) test. The 2 simulations were performed using Molecular Operating Environment (MOE) 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356) based on ΔG binding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.

  18. Modification of -Adenosyl--Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

    Directory of Open Access Journals (Sweden)

    Usman Sumo Friend Tambunan

    2017-04-01

    Full Text Available Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5 methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S -adenosyl- l -methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S -adenosyl- l -homocysteine (SAH. The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity test. The 2 simulations were performed using Molecular Operating Environment (MOE 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356 based on ΔG binding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.

  19. Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

    Directory of Open Access Journals (Sweden)

    Dong-Hoon Chung

    2014-06-01

    Full Text Available Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV, a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM, for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

  20. 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.

  1. Expression and Stability of Foreign Epitopes Introduced into 3A Nonstructural Protein of Foot-and-Mouth Disease Virus

    Science.gov (United States)

    Li, Pinghua; Bai, Xingwen; Cao, Yimei; Han, Chenghao; Lu, Zengjun; Sun, Pu; Yin, Hong; Liu, Zaixin

    2012-01-01

    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. PMID:22848509

  2. Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA Vaccine.

    Science.gov (United States)

    Bao, Huihui; Ramanathan, Aarti A; Kawalakar, Omkar; Sundaram, Senthil G; Tingey, Colleen; Bian, Charoran B; Muruganandam, Nagarajan; Vijayachari, Paluru; Sardesai, Niranjan Y; Weiner, David B; Ugen, Kenneth E; Muthumani, Karuppiah

    2013-02-01

    Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

  3. The use of non-structural proteins of foot and mouth disease virus (FMDV) to differentiate between vaccinated and infected animals

    International Nuclear Information System (INIS)

    2007-05-01

    The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture has a long history of coordinating isotope aided research projects for improving animal productivity in developing countries. Foot and mouth disease (FMD) remains a tremendous problem in developing countries and is a constant threat to developed countries. Tests to determine the immune status of animals form the basis of understanding the control of the disease. Vaccination is widely employed and has to be on a continuous basis. The antibodies produced against the FMD virus (FMDV) after infection are the same as those produced on vaccination. However, tests have been devised to use non-structural proteins (NSP) of FMDV since it is only on infection that antibodies are produced against such proteins. Thus, through their specific detection, it is possible to determine whether animals are infected in the face of vaccination. This is important since any contact with replicating virus in cattle, sheep and goats may result in a non-clinical situation where virus is carried by the affected animal without symptoms, and may be a threat to others. There is great suspicion over animals where virus has multiplied and so their identification is paramount and essential where countries are trying to demonstrate virus freedom. There have been many developments in this field and the IAEA sought to try and validate methods in this coordinated research project (CRP). Validation per se is always addressed by the IAEA and they have been instrumental in improving guidelines for test certification through the OIE. Although FMD tests had been devised they were not fully examined in a large geographical spread, nor were they compared directly. During the CRP many variations of tests were produced and this complicated the validation process. The resulting TECDOC reflects the relative instability of developments but value adds to the latest opinions on the use of NSP tests in the control of FMD. Several commercial kits

  4. Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan.

    Science.gov (United States)

    Chen, Tsu-Han; Lee, Fan; Lin, Yeou-Liang; Pan, Chu-Hsiang; Shih, Chia-Ni; Tseng, Chun-Hsien; Tsai, Hsiang-Jung

    2016-04-01

    Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus. Copyright © 2014. Published by Elsevier B.V.

  5. Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

    Directory of Open Access Journals (Sweden)

    Thomas Wurster

    Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  6. Elevated Dengue Virus Nonstructural Protein 1 Serum Levels and Altered Toll-Like Receptor 4 Expression, Nitric Oxide, and Tumor Necrosis Factor Alpha Production in Dengue Hemorrhagic Fever Patients

    Directory of Open Access Journals (Sweden)

    Denise Maciel Carvalho

    2014-01-01

    Full Text Available Background. During dengue virus (DV infection, monocytes produce tumor necrosis factor alpha (TNF-α and nitric oxide (NO which might be critical to immunopathogenesis. Since intensity of DV replication may determine clinical outcomes, it is important to know the effects of viral nonstructural protein 1 (NS1 on innate immune parameters of infected patients. The present study investigates the relationships between dengue virus nonstructural protein 1 (NS1 serum levels and innate immune response (TLR4 expression and TNF-α/NO production of DV infected patients presenting different clinical outcomes. Methodology/Principal Findings. We evaluated NO, NS1 serum levels (ELISA, TNF-α production by peripheral blood mononuclear cells (PBMCs, and TLR4 expression on CD14+ cells from 37 dengue patients and 20 healthy controls. Early in infection, increased expression of TLR4 in monocytes of patients with dengue fever (DF was detected compared to patients with dengue hemorrhagic fever (DHF. Moreover, PBMCs of DHF patients showed higher NS1 and lower NO serum levels during the acute febrile phase and a reduced response to TLR4 stimulation by LPS (with a reduced TNF-α production when compared to DF patients. Conclusions/Significance. During DV infection in humans, some innate immune parameters change, depending on the NS1 serum levels, and phase and severity of the disease which may contribute to development of different clinical outcomes.

  7. Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non-structural Triple Gene Block Protein 1 and Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Dědič, P.

    2012-01-01

    Roč. 160, č. 5 (2012), s. 251-254 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus M * recombinant protein * coat protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.000, year: 2012

  8. Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2017-02-01

    Full Text Available Porcine Reproductive and Respiratory Syndrome (PRRS is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV. PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5 region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs and peripheral blood monocytes (PBMCs were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

  9. Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

    Science.gov (United States)

    McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

    2017-07-11

    The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  11. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    International Nuclear Information System (INIS)

    Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

    2017-01-01

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine arteritis virus.

  12. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2017-05-15

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine

  13. Heat shock protein 90 positively regulates Chikungunya virus replication by stabilizing viral non-structural protein nsP2 during infection.

    Directory of Open Access Journals (Sweden)

    Indrani Das

    Full Text Available BACKGROUND: The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV emphasize the need to understand the biology of the virus for developing effective antiviral therapies. METHODS AND FINDINGS: In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90 mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27 and Indian outbreak strain of 2006 (DRDE-06. Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. CONCLUSION: Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06

  14. Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

    Science.gov (United States)

    Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

    2002-07-01

    The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

  15. Comparison of 2 commercial single-dose Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV) vaccines on pigs dually infected with M. hyopneumoniae and PRRSV.

    Science.gov (United States)

    Park, Changhoon; Kang, Ikjae; Seo, Hwi Won; Jeong, Jiwoon; Choi, Kyuhyung; Chae, Chanhee

    2016-04-01

    The objective of this study was to compare the efficacy of 2 different commercial Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV) vaccines in regard to growth performance, microbiological and immunological analyses, and pathological observation from wean to finish (175 d of age). Pigs were administered M. hyopneumoniae and PRRSV vaccines at 7 and 21 d of age, respectively, or both at 21 d old and then challenged with both M. hyopneumoniae and PRRSV at 49 d old. Significant (P hyopneumoniae, M. hyopneumoniae-specific interferon-γ secreting cells, and macroscopic and microscopic lung lesions. Induction of interleukin-10 following PRRSV vaccination does not interfere with the immune responses induced by M. hyopneumoniae vaccine. The present study demonstrated that the single-dose vaccination regimen for M. hyopneumoniae and PRRSV vaccine is efficacious for controlling coinfection with M. hyopneumoniae and PRRSV based on clinical, microbiological, immunological, and pathological evaluation.

  16. Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

    Science.gov (United States)

    Jääskeläinen, Kirsi M; Plyusnina, Angelina; Lundkvist, Ake; Vaheri, Antti; Plyusnin, Alexander

    2008-01-11

    The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

  17. Udvikling af antistoffer efter vaccination mod og podning med PRRSV

    DEFF Research Database (Denmark)

    Sonne Kristensen, Charlotte; Qvist Pawlowski, Mia; Thoning, Henrik

    konkludere, at det er muligt at vaccinere med begge PRRS vacciner på samme tid og opnå antistofsvar for begge typer PRRSV. Husk dog altid at give forskellige vacciner i hver sin side af nakken. Blodprøver fra grisene viste, at de havde dannet antistoffer og blev positive i ELISA-testen for PRRSV Type 1 21......-US samt IPT testene. Disse tests kan dog ikke skelne ”eksotiske” PRRSV subtyper fra de almindelige cirkulerende PRRSV subtyper i Danmark. Forsøget blev gennemført på Lindholm. Der indgik 66 grise fra en Blå SPF+Ap6+Ap12 besætning. Grisene blev ved ankomst fordelt i 4 grupper (VAC-T1, VAC-T2, VAC-T1T2 og...... NON-VAC): VAC-T1 blev vaccineret med Porcilis ® PRRS VET VAC-T2 blev vaccineret med Ingelvac ® PRRS VET VAC-T1T2 fik begge vacciner samtidig i hver sin side af nakken NON-VAC blev ikke vaccineret. Grisene blev gennem hele forsøget observeret dagligt. De fik taget temperatur den første uge efter...

  18. PRRSV outbreak with high mortality in northern part of Denmark

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine; Hjulsager, Charlotte Kristiane; Rathkjen, P. H.

    with high mortality rate in piglets occurred in Northern Jutland. PRRSV type 2 was detected by real-time RT-PCR in lung tissue from 10 days old piglets. The outbreak was treated by extensive vaccination with Ingelvac® PRRS MLV and strict management procedures. 6 weeks later, the mortality of liveborn...

  19. Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

    Science.gov (United States)

    Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

    2013-01-01

    Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

  20. The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

    Science.gov (United States)

    Kenney, Scott P.

    2015-01-01

    hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future. PMID:25609799

  1. Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

    DEFF Research Database (Denmark)

    Oleksiewicz, Martin B.; Nielsen, Jens

    1999-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely...... infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages....

  2. The Development of Nonstructural Alternatives.

    Science.gov (United States)

    1979-05-01

    particular appeal and benefits for those concerned with environmental issues. However, on reflection it is clear that, almost by definition, easily...peculiar to nonstructural alternatives (that is, the social costs are borne primaqrilv by th,, )ent ficiaries) makes them appealing from the national...Costs Of prId-nt ! loo41p l jotve iopment lie basically in the costs of elevated 111’Z ’’l’i. Pie 1land reqat reti c,- snob dovelIopmeot is loss

  3. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine.

    Directory of Open Access Journals (Sweden)

    Yuanfang Fu

    Full Text Available Foot-and-mouth disease (FMD is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab was found to recognize a conserved "AEKNPLE" epitope spanning amino acids 109-115 of non-structural protein (NSP 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain, which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant "AEKNPLE" epitope in NSP 3A of FMDV.

  4. Expression of the rice hoja blanca virus (RHBV non-structural protein 3 (NS3 in Escherichia coli and its in situ localization in RHBV-infected rice tissues

    Directory of Open Access Journals (Sweden)

    Miguel Muñoz

    2004-09-01

    Full Text Available The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV was fused to the glutathione- S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein. Rev. Biol. Trop. 52(3: 765-775. Epub 2004 Dic 15.El gen que codifica por la proteína no estructural NS3 del virus de la hoja blanca de arroz (RHBV se fusionó al extremo carboxilo del gen de la glutationa-S-transferasa y se expresó en la cepa JM83 de Escherichia coli. Se obtuvieron altas concentraciones de la proteína de fusion (GST-NS3 en forma insoluble. La proteína de fusión se fraccionó en geles de SDS-PAGE, se purificó por electroelución, y se utilizó para producir anticuerpos policlonales en conejo . El antisuero producido se absorbió con extractos crudos de E. coli. Extractos crudos de plantas de arroz sanas e infectadas con el RHBV se evaluaron por Western blots detectándose una banda de peso molecular similar al estimado para la proteína NS3 (23KDa en las plantas infectadas con el virus. Los tejidos provenientes de plantas infectadas con el RHBV se analizaron por medio de microscopia inmunoelectrónica con oro colloidal marcado con anticuerpos contra la proteína NS3 y la nucleoproteína viral N. Se observó una acumulación in situ de la

  5. Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

    Science.gov (United States)

    Reyes-Ruiz, José M; Osuna-Ramos, Juan F; Cervantes-Salazar, Margot; Lagunes Guillen, Anel E; Chávez-Munguía, Bibiana; Salas-Benito, Juan S; Del Ángel, Rosa M

    2018-02-01

    Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Directory of Open Access Journals (Sweden)

    Sylvaine Boissinot

    Full Text Available Cucurbit aphid-borne yellows virus (CABYV is a polerovirus (Luteoviridae family with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT. Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT* incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  7. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Science.gov (United States)

    Boissinot, Sylvaine; Erdinger, Monique; Monsion, Baptiste; Ziegler-Graff, Véronique; Brault, Véronique

    2014-01-01

    Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  8. Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

    Science.gov (United States)

    Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

    2013-12-01

    Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus

    Czech Academy of Sciences Publication Activity Database

    Pečenková, Tamara; Filigarová, Marie; Čeřovská, Noemi

    2005-01-01

    Roč. 41, - (2005), s. 128-135 ISSN 1046-5928 R&D Projects: GA ČR GA522/04/1329 Institutional research plan: CEZ:AV0Z50380511 Keywords : Protein expression * Potato mop-top virus * Triple gene block Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.553, year: 2005

  10. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    Science.gov (United States)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  11. Prediction and in vitro verification of potential CTL epitopes conserved among PRRSV-2 strains

    DEFF Research Database (Denmark)

    Welner, Simon; Nielsen, Morten; Rasmussen, Michael

    2017-01-01

    Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the causative agent of one of the most important porcine diseases with a high impact on animal health, welfare, and production economy. PRRSV exhibits a multitude of immunoevasive strategies that, in combination with a very high...

  12. Protection against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection through Passive Transfer of PRRSV-Neutralizing Antibodies Is Dose Dependent▿ †

    OpenAIRE

    Lopez, O. J.; Oliveira, M. F.; Garcia, E. Alvarez; Kwon, B. J.; Doster, A.; Osorio, F. A.

    2007-01-01

    Previous work in our laboratory demonstrated that passive transfer of porcine reproductive and respiratory syndrome virus (PRRSV)-neutralizing antibodies (NA) protected pregnant sows against reproductive failure and conferred sterilizing immunity in sows and offspring. We report here on the dose requirement for protection by passive transfer with NA in young weaned pigs. The presence of a 1:8 titer of PRRSV-NA in serum consistently protected pigs against viremia. Nevertheless, their lungs, to...

  13. Real-life prevalence of resistance-associated variants against non-structural protein 5A inhibitors and efficiency of Daclatasvir + Asunaprevir therapy in Korean patients with genotype 1b hepatitis C.

    Science.gov (United States)

    Yu, Jung Hwan; Lee, Jung Il; Lee, Kwan Sik; Kim, Ja Kyung

    2017-08-24

    Direct-acting antivirals (DAAs) for chronic hepatitis C (CHC) treatment are tolerable and highly effective in a shorter period of time than before. However, resistance-associated variants (RAVs) can affect the efficacy of DAAs. The aim of this study was to investigate the real-life prevalence of RAVs against non-structural protein 5A (NS5A) inhibitors in Korean patients with genotype 1b chronic hepatitis C. All consecutive patients with CHC genotype 1b who underwent a RAV test at a single referral hospital were enrolled. A total of 142 patients (male 53, female 89) were tested for RAVs. The average age of the patients was 58 years. Liver cirrhosis was found in 34.5% (49/142) of patients, and 19.0% (29/142) of patients had previously undergone interferon-based treatment. Twenty-nine patients (20.4%) had RAVs (Y93 or L31). Y93H, L31, or Y93H with L31 were detected in 22 (15.5%), 8 (5.6%), and 1 (0.7%) patients, respectively. The presence of RAV was not affected by previous interferon-based treatment or by the existence of liver cirrhosis. Among 113 patients without baseline NS5A RAVs, 72 patients started daclatasvir (DCV) + asunaprevir (ASV) treatment and 95% (68/72) patients achieved virologic response at week 4. Virologic response at end of treatment and sustained virologic response at 12 weeks after treatment were achieved by 94% (68/72) and 94% (68/72), respectively. In Korean patients with genotype 1b CHC, 20.4% (29 of 142) of patients showed RAVs against NS5A inhibitors. Patient without RAVs who received treatment with DCV + ASV showed high virologic response rates in Korea.

  14. Laser-cut paper-based device for the detection of dengue non-structural NS1 protein and specific IgM in human samples.

    Science.gov (United States)

    Theillet, G; Rubens, A; Foucault, F; Dalbon, P; Rozand, C; Leparc-Goffart, I; Bedin, F

    2018-03-10

    The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical areas worldwide, affecting hundreds of millions of people each year. Dengue viruses are typically transmitted by mosquitoes and can cause a wide range of symptoms from flu-like fever to organ impairment and death. Although conventional diagnostic tests can provide early diagnosis of acute dengue infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop affordable, simple, rapid, and robust diagnostic tools that can be used at 'Point of Care' settings. Early diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, a novel laser-cut device made of glass-fiber paper was designed and tested for the detection of the dengue Non Structural 1 (NS1) viral protein and specific IgM in blood and plasma. The device, called PAD, was able to detect around 25 ng/mL of NS1 protein in various sample types in 8 minutes, following a few simple steps. The PAD was also able to detect specific IgM in human plasmas in less than 10 minutes. The PAD appears to have all the potential to assist health workers in early diagnosis of dengue fever or other tropical fevers caused by flaviviruses.

  15. Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1).

    Science.gov (United States)

    Gelanew, Tesfaye; Hunsperger, Elizabeth

    2018-02-06

    Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.

  16. Hemagglutinating virus of Japan envelope (HVJ-E) can enhance the immune responses of swine immunized with killed PRRSV vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Zhihong [State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); China Institute of Veterinary Drug Control, Beijing 100081 (China); Zhang, Quan [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Wang, Zaishi [China Institute of Veterinary Drug Control, Beijing 100081 (China); Zhang, Zhongqiu [State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Veterinary Bureau, Ministry of Agriculture of the People' s Republic of China, Beijing 100125 (China); Guo, Pengju [Institute of Veterinary Medicine, Guangdong Academy of Agricultural Sciences, Guangdong 510640 (China); Zhao, Deming, E-mail: zhaodm@cau.edu.cn [State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We investigated the immunoadjuvant effects of HVJ-E on killed PRRSV vaccine. Black-Right-Pointing-Pointer HVJ-E enhanced the humoral and cellular responses of the piglets to PRRSV. Black-Right-Pointing-Pointer It is suggested that HVJ-E could be developed as a new-type adjuvant for mammals. -- Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically detrimental pig pathogen that causes significant losses for the pig industry. The immunostimulatory effects of hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy and the adjuvant efficacy of HVJ-E have been previously evaluated. The objective of this study was to investigate the adjuvant effects of HVJ-E on immunization with killed PRRSV vaccine, and to evaluate the protective effects of this immunization strategy against virulent PRRSV infection in piglets. Next, the PRRSV-specific antibody response, lymphocyte proliferation, PRRSV-specific IL-2, IL-10 and IFN-{gamma} production, and the overall protection efficacy were evaluated to assess the immune responses of the piglets. The results showed that the piglets inoculated simultaneously with killed PRRSV vaccine and HVJ-E had a significantly stronger immune response than those inoculated with killed PRRSV vaccine alone. Our results suggest that HVJ-E could be employed as an effective adjuvant to enhance the humoral and cellular responses of piglets to PRRSV.

  17. Complete Genome Sequence of a Recombinant NADC30-Like Strain, SCnj16, of Porcine Reproductive and Respiratory Syndrome Virus in Southwestern China

    Science.gov (United States)

    Kang, Runmin; Xie, Bo; Tian, Yiming; Yang, Xin; Yu, Jifeng

    2018-01-01

    ABSTRACT The NADC30-like strains of porcine reproductive and respiratory syndrome virus (PRRSV) are characterized by a 131-amino-acid deletion in nonstructural protein 2 (NSP2). Here, we report the complete genome sequence of a recombinant NADC30-like PRRSV strain, SCnj16, that exhibits the molecular marker of the Chinese highly pathogenic PRRSV (HP-PRRSV) in NSP2. PMID:29439029

  18. Porcine reproductive and respiratory syndrome virus (PRRSV): pathogenesis and interaction with the immune System

    Science.gov (United States)

    This review addresses important issues of porcine reproductive and respiratory syndrome virus (PRRSV) infection, immunity, pathogenesis and control. Worldwide PRRS is the most economically important infectious disease of pigs. We highlight the latest information on viral genome structure, pathogenic...

  19. Epidemiological study of air filtration systems for preventing PRRSV infection in large sow herds.

    Science.gov (United States)

    Alonso, Carmen; Murtaugh, Michael P; Dee, Scott A; Davies, Peter R

    2013-10-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant pathogen in the US swine industry. Aerosol transmission among herds is a major concern in pig dense regions and filtration of incoming air, in combination with standard biosecurity procedures, has been demonstrated to prevent transmission of PRRSV into susceptible herds. To quantify the impact of air filtration on reducing risk of PRRSV outbreaks, we compared the incidence rate of new PRRSV introductions in 20 filtered and 17 non-filtered control sow herds in a swine dense region of North America during a 7 year study period. Events of novel virus introduction were ascertained by phylogenetic analysis of PRRSV ORF5 gene sequences. Putative new viruses were defined as exogenous (introduced) based on ORF5 nucleotide sequence differences compared to previous farm isolates. The influence of sequence difference cut-off values ranging from 2 to 10% on case definition and relative risk were evaluated. Non-filtered farms incurred about 0.5 outbreaks per year, with a seasonal increase in risk in cooler periods. Baseline risk, prior to filtration, in treatment farms was approximately 0.75 per year, approximately 50% higher than in control farms. Air filtration significantly reduced risk of PRRSV introduction events to 0.06-0.22 outbreaks per year, depending on the cut-off values used to classify a virus isolate as new to the herd. Overall, air filtration led to an approximately 80% reduction in risk of introduction of novel PRRSV, indicating that on large sow farms with good biosecurity in swine-dense regions, approximately four-fifths of PRRSV outbreaks may be attributable to aerosol transmission. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

    Directory of Open Access Journals (Sweden)

    Chungwon J Chung

    Full Text Available Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI. However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates.An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983 and another strain (type-2 PRRSVVR2332 with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection.At 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate.These results demonstrated that T-lymphocytes recognizing antigenically and

  1. Accumulation pattern of total nonstructural carbohydrate in ...

    African Journals Online (AJOL)

    Umukoro

    1977-09-09

    Sep 9, 1977 ... 1Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Famaillá, Argentina. 2Department of Plant Sciences, University of California Davis, CA, USA. Accepted 17 October, 2012. The pattern of total nonstructural carbohydrate (TNC) accumulation in strawberry (Fragaria ananassa. Duch.) nursery ...

  2. Accumulation pattern of total nonstructural carbohydrate in ...

    African Journals Online (AJOL)

    The pattern of total nonstructural carbohydrate (TNC) accumulation in strawberry (Fragaria ananassa Duch.) nursery runner plants, cv. eCamarosaf, was determined for three growing seasons. Plant growth and fruit production patterns were also evaluated. The experiments were carried out on plants propagated in high ...

  3. Chinese and Vietnamese strains of HP-PRRSV cause different pathogenic outcomes in United States high health swine

    Science.gov (United States)

    An infectious clone of a highly pathogenic PRRSV strain from Vietnam (rSRV07) was prepared, analyzed and compared to Chinese highly pathogenic PRRSV rJXwn06 and US Type 2 prototype VR-2332 in order to examine the effects of virus phenotype and genotype on growth in MARC-145 cells, as well as the imp...

  4. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    International Nuclear Information System (INIS)

    Lee, Changhee; Yoo, Dongwan

    2006-01-01

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-ΔE-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-ΔE virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-ΔE virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm

  5. Cellular immune responses in the lungs of pigs infected in utero with PRRSV: An immunohistochemical study

    DEFF Research Database (Denmark)

    Tingstedt, Jens Erik; Nielsen, Jens

    2004-01-01

    The cellular response in the lungs of pigs transplacentally infected with porcine reproductive and respiratory syndrome virus (PRRSV) was examined by immunohistochemistry. Double staining for the T-cell marker antigen CD3 and PRRSV demonstrated that the appearance and distribution of T-cells homing...... to the lungs of infected pigs correlated well with the presence and location of virus-infected cells. Single stainings showed that cells positive for the CD2 and CD8 antigen were almost as numerous in pneumonic lesions as CD3 positive cells whereas cells expressing the CD4 antigen were rare. The morphology...

  6. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

    2011-01-01

    to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with ¿40 or ¿25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP¿40 acquired various...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

  7. Detection of PRRSV in 218 field samples using six molecular methods: What we are looking for?

    DEFF Research Database (Denmark)

    Toplak, Ivan; Štukelj, Marina; Gracieux, Patrice

    2012-01-01

    on the genetic make-up of the target viruses and confirm findings of a previous study where we showed some commercial PCR kits failed to detect specific genetic linkages of PRRSV. Thus, these finding emphatise that it is cricial that the manifactors ofl diagnostic PCR kits (conventional and real...

  8. Temporal evolution and potential recombination events in PRRSV strains of Sonora Mexico.

    Science.gov (United States)

    Burgara-Estrella, Alexel; Reséndiz-Sandoval, Mónica; Cortey, Martí; Mateu, Enric; Hernández, Jesús

    2014-12-05

    The aim of this work was to examine the evolution and potential existence of intragenic recombinations of PRRSV strains in Sonora, Mexico. In this study, 142 serum samples from farms located in Hermosillo (HMO), Cd. Obregón (OBR) and Navojoa (NAV) were sequenced from 2002 to 2012. Ninety non-redundant sequences of ORF5 gene were analyzed for temporal and spatial relationships among strains and the probability of a recombination event. The phylogenetic analysis showed 30 strains grouped into eight groups; 16 strains were closely related among the farms, while 14 were un-related. The first strain in this study was observed in 2002. A number of farms were infected with one or more strains, and in the majority of the strains, the virus was replaced by a new strain. The recombination analysis suggested the presence of four viruses as products of a recombination event; in one case, a virus close related with MLV vaccine was involved as the parent virus. This work shows the evolution of PRRSV in the field, the viral dissemination between farms and the potential recombination events. Our data suggest that PRRSV in Sonora has a specific genetic nature compared with other PRRSV. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Fulminant sepsis is a cardinal sign of HP-PRRSV in pigs

    Science.gov (United States)

    In 2006 a unique syndrome with high morbidity and mortality was recognized in growing pigs in China that became known as porcine high fever disease (PHFD). One consistent finding in affected pigs was the detection of porcine reproductive and respiratory syndrome virus (PRRSV) that had unique nsp2 ge...

  10. Experimental infection of pigs with two East European variants of Type 1 PRRSV

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik; Heegaard, Peter M. H.

    Porcine reproductive and respiratory syndrome viruses (PRRSV) have been divided into Type 1 (European) and Type 2 (North American) viruses. PRRSV are very diverse and Type 1 viruses have even been further divided into subtypes. While Type 1 viruses from Western Europe belong to subtype 1, viruses...... the subtype 1 strains. The aim of this project was to study the infection dynamics and clinical and pathological impact of two east European Type 1 strains. In an experimental trial, infection of pigs with the Russian subtype 2 strain “Ili6” and the Belarusian atypical isolate “Bor59” were compared...... to an early “Lelystad-like” Danish subtype 1 isolate “18794”. Groups of seven pigs of unique high sanitary status were infected with one of the three PRRSV isolates, and a fourth group served as sham-inoculated controls. The pigs were monitored for 24 days, and nasal swabs and blood samples were taken at 0, 3...

  11. Probing genetic control of swine responses to PRRSV infection: current progress of the PRRS host genetics consortium

    Directory of Open Access Journals (Sweden)

    Lunney Joan K

    2011-06-01

    Full Text Available Abstract Background Understanding the role of host genetics in resistance to porcine reproductive and respiratory syndrome virus (PRRSV infection, and the effects of PRRS on pig health and related growth, are goals of the PRRS Host Genetics Consortium (PHGC. Methods The project uses a nursery pig model to assess pig resistance/susceptibility to primary PRRSV infection. To date, 6 groups of 200 crossbred pigs from high health farms were donated by commercial sources. After acclimation, the pigs were infected with PRRSV in a biosecure facility and followed for 42 days post infection (dpi. Blood samples were collected at 0, 4, 7, 10, 14, 21, 28, 35 and 42 dpi for serum and whole blood RNA gene expression analyses; weekly weights were recorded for growth traits. All data have been entered into the PHGC relational database. Genomic DNAs from all PHGC1-6 pigs were prepared and genotyped with the Porcine SNP60 SNPchip. Results Results have affirmed that all challenged pigs become PRRSV infected with peak viremia being observed between 4-21 dpi. Multivariate statistical analyses of viral load and weight data have identified PHGC pigs in different virus/weight categories. Sera are now being compared for factors involved in recovery from infection, including speed of response and levels of immune cytokines. Genome-wide association studies (GWAS are underway to identify genes and chromosomal locations that identify PRRS resistant/susceptible pigs and pigs able to maintain growth while infected with PRRSV. Conclusions Overall, the PHGC project will enable researchers to discover and verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection. The availability of PHGC samples provides a unique opportunity to continue to develop deeper phenotypes on every PRRSV infected pig.

  12. Metabolism of non-structural carbohydrates in ruminants

    OpenAIRE

    Cañizares, G. I L [UNESP; Rodrigues, L. [UNESP; Cañizares, M. C. [UNESP

    2009-01-01

    The carbohydrates provide 50 to 80% of the dry matter of grain and roughage and can be divided into structural (cellulose, hemicellulose) and non-structural (starch, pectin and sugars). The non-structural carbohydrates are primarily digested in the rumen and its dynamic process is a sequence for the supply of nutrients to the intestine. The quality and quantity of products resulting from ruminal fermentation are dependent on the type and activity of microorganisms in the rumen influenced by t...

  13. Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection.

    Science.gov (United States)

    Fleming, Damarius S; Miller, Laura C

    2018-04-01

    It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs have emerged as having an important role in the immune system in humans. The study uses transcriptomic read counts to profile the type and quantity of both well and lesser characterized sncRNAs, such as microRNAs and small nucleolar RNAs to identify and quantify the classes of sncRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs. Our results returned evidence on nine classes of sncRNA, four of which were consistently statistically significantly different based on Fisher's Exact Test, that can be detected and possibly interrogated for their effect on host dysregulation during PRRSV infections. Published by Elsevier Inc.

  14. Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection

    Science.gov (United States)

    It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs ...

  15. Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived cells at differential activation statuses

    Science.gov (United States)

    Activation statuses of monocytic cells are critically important for antiviral immunity. Devastating viruses like porcine reproductive and respiratory syndrome virus (PRRSV) are capable of directly infecting these cells, subverting host immunity. Monocyte-derived DCs (mDCs) are major target cells in ...

  16. Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain.

    Science.gov (United States)

    Schlink, Sarah N; Lager, Kelly M; Brockmeier, Susan L; Loving, Crystal L; Miller, Laura C; Vorwald, Ann C; Yang, Han-Chun; Kehrli, Marcus E; Faaberg, Kay S

    2016-10-15

    Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for one of the most economically important diseases in swine worldwide. It causes reproductive failure in sows and pneumonia in pigs that predisposes them to secondary bacterial infections. Methods to control PRRSV and/or limit secondary bacterial infections are desired to reduce the impact of this virus on animal health. Neutrophils play a major role in combatting infection; they can act as phagocytes as well as produce and release lytic enzymes that have potent antimicrobial effects leading to the destruction and clearance of bacterial pathogens. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that controls the production, differentiation and function of granulocytes (including neutrophils) from the bone marrow. Recent work from our laboratory has shown that encoding porcine G-CSF in a replication-defective adenovirus (Ad5-G-CSF) and delivering a single dose to pigs induced a neutrophilia lasting more than two weeks. As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with highly pathogenic (HP)-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection. Administration of Ad5-G-CSF induced a significant neutrophilia as expected. However, between 1 and 2days following HP-PRRSV challenge the number of circulating neutrophils decreased dramatically in the HP-PRRSV infected group, but not the non-infected Ad5-G-CSF group. Ad5-G-CSF administration induced monocytosis as well, which was also reduced by HP-PRRSV challenge. There was no difference in the progression of disease between the Ad5-G-CSF and Ad5-empty groups following HP-PRRSV challenge, with pneumonia and systemic bacterial infection occurring in both treatment groups. Given the impact of HP-PRRSV infection on the

  17. Non-structural Components influencing Hospital Disaster Preparedness in Malaysia

    Science.gov (United States)

    Samsuddin, N. M.; Takim, R.; Nawawi, A. H.; Rosman, M. R.; SyedAlwee, S. N. A.

    2018-04-01

    Hospital disaster preparedness refers to measures taken by the hospital’s stakeholders to prepare, reduce the effects of disaster and ensure effective coordination during incident response. Among the measures, non-structural components (i.e., medical laboratory equipment & supplies; architectural; critical lifeline; external; updated building document; and equipment & furnishing) are critical towards hospital disaster preparedness. Nevertheless, over the past few years these components are badly affected due to various types of disasters. Hence, the objective of this paper is to investigate the non-structural components influencing hospital’s disaster preparedness. Cross-sectional survey was conducted among thirty-one (31) Malaysian hospital’s employees. A total of 6 main constructs with 107 non-structural components were analysed and ranked by using SPSS and Relative Importance Index (RII). The results revealed that 6 main constructs (i.e. medical laboratory equipment & supplies; architectural; critical lifeline; external; updated building document; and equipment & furnishing) are rated as ‘very critical’ by the respondents. Among others, availability of medical laboratory equipment and supplies for diagnostic and equipment was ranked first. The results could serve as indicators for the public hospitals to improve its disaster preparedness in terms of planning, organising, knowledge training, equipment, exercising, evaluating and corrective actions through non-structural components.

  18. Establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with PRRSV-1

    Directory of Open Access Journals (Sweden)

    Helen eSingleton

    2016-06-01

    Full Text Available Monocyte-derived macrophages (MoMØ and monocyte-derived dendritic cells (MoDC are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV is known to infect myeloid cells, such as macrophages (MØ and dendritic cells (DC. Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated monocyte-derived macrophages (MoMØ were stimulated with activators for classical (M1 or alternative (M2 activation. GM-CSF and IL-4 generated monocyte-derived dendritic cells (MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells towards PRRSV-1 infection.

  19. Nonstructural Proteins of Alphavirus—Potential Targets for Drug Development

    Directory of Open Access Journals (Sweden)

    Farhana Abu Bakar

    2018-02-01

    Full Text Available Alphaviruses are enveloped, positive single-stranded RNA viruses, typically transmitted by arthropods. They often cause arthralgia or encephalitic diseases in infected humans and there is currently no targeted antiviral treatment available. The re-emergence of alphaviruses in Asia, Europe, and the Americas over the last decade, including chikungunya and o’nyong’nyong viruses, have intensified the search for selective inhibitors. In this review, we highlight key molecular determinants within the alphavirus replication complex that have been identified as viral targets, focusing on their structure and functionality in viral dissemination. We also summarize recent structural data of these viral targets and discuss how these could serve as templates to facilitate structure-based drug design and development of small molecule inhibitors.

  20. Comparative analysis of signature genes in porcine reproductive and respiratory syndrome virus (PRRSV)-infected porcine monocyte-derived dendritic cells at differential activation statuses

    Science.gov (United States)

    Activation statuses of monocytic cells, e.g. monocytes, macrophages and dendritic cells (DCs), are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cell...

  1. Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

    Science.gov (United States)

    Steinrigl, Adolf; Revilla-Fernández, Sandra; Wodak, Eveline; Schmoll, Friedrich; Sattler, Tatjana

    2014-01-01

    Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid.

  2. Identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain.

    Science.gov (United States)

    Sun, Haiyan; Pattnaik, Asit K; Osorio, Fernando A; Vu, Hiep L X

    2016-12-01

    We recently generated a fully synthetic porcine reproductive and respiratory syndrome virus strain (designated as PRRSV-CON), which confers unprecedented levels of heterologous protection. We report herein that the synthetic PRRSV-CON possesses a unique phenotype in that it induces type-I interferons (IFNs) instead of suppressing these cytokines as most of the naturally occurring PRRSV isolates do. Through gain- and loss- of-function studies, the IFN-inducing phenotype of PRRSV-CON was mapped to the 3.3kb genomic fragment encoding three viral nonstructural proteins: nsp1α, nsp1β and the N-terminal part of nsp2. Further studies indicated that a cooperation among these 3 proteins was required for effective induction of IFNs. Collectively, this study constitutes the first step toward understanding the mechanisms by which the synthetic PRRSV-CON confers heterologous protection. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. A Non-Structural Investigation of VIX Risk Neutral Density

    DEFF Research Database (Denmark)

    Barletta, Andrea; Santucci de Magistris, Paolo; Violante, Francesco

    We propose a non-structural pricing method to derive the risk-neutral density (RND) implied by options on the CBOE Volatility Index (VIX). The methodology is based on orthogonal polynomial expansions around a kernel density and yields the RND of the underlying asset without the need for a paramet......We propose a non-structural pricing method to derive the risk-neutral density (RND) implied by options on the CBOE Volatility Index (VIX). The methodology is based on orthogonal polynomial expansions around a kernel density and yields the RND of the underlying asset without the need...... for a parametric specification. The classic family of Laguerre expansions is extended to include the GIG and the generalized Weibull kernels, thus relaxing the conditions required on the tail decay rate of the RND to ensure convergence. We show that the proposed methodology yields an accurate approximation...... of the RND in a large variety of cases, also when the no-arbitrage and efficient option prices are contaminated by measurement errors. Our empirical investigation, based on a panel of traded VIX options, reveals some stylized facts on the RND of VIX. We find that a common stochastic factor drives the dynamic...

  4. Genetic and biological characterization of a Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2)causing significant clinical disease in the field

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine; Larsen, Lars Erik; Hjulsager, Charlotte Kristiane

    2017-01-01

    pathogenic or vaccine evading PRRSV strain had emerged in Denmark. The overall aim of the present study was to perform a genetic and biological characterization of the virus isolated from the diseased herd. Complete genome sequencing of isolates from this herd revealed that although the case strain had some...... in the Northern part of Denmark experienced an infection with PRRSV-2 with clinical signs that were much more severe than normally reported from current Danish PRRSV-2 affected herds. Due to the clinical observations of reproductive failure in sows and high mortality in piglets, it was speculated that a new, more...... unique genetic features including a deduced 3 amino acid deletion, it was in overall very similar to the other PRRS-2 viruses circulating in Denmark. In an experimental trial in growing pigs, no overt clinical signs or pathology were observed following intranasal inoculation with the new virus isolate...

  5. Seasonal dynamics and age of stemwood nonstructural carbohydrates in temperate forest trees

    Science.gov (United States)

    Andrew D. Richardson; Mariah S. Carbone; Trevor F. Keenan; Claudia I. Czimczik; David Y. Hollinger; Paula Murakami; Paul G. Schaberg; Xiaomei. Xu

    2013-01-01

    Nonstructural carbohydrate reserves support tree metabolism and growth when current photosynthates are insufficient, offering resilience in times of stress. We monitored stemwood nonstructural carbohydrate (starch and sugars) concentrations of the dominant tree species at three sites in the northeastern United States. We estimated the mean age of the starch and sugars...

  6. NIRS determination of non-structural carbohydrates, water soluble carbohydrates and other nutritive quality traits in whole plant maize with wide range variability

    OpenAIRE

    L. Campo; A. B. Monteagudo; B. Salleres; P. Castro; J. Moreno-Gonzalez

    2013-01-01

    The aim of this work was to study the potential of near-infrared reflectance spectroscopy (NIRS) to predict non-structural carbohydrates (NSC), water soluble carbohydrates (WSC), in vitro organic dry matter digestibility (IVOMD), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and starch in samples of whole plant maize with a wide range of variability. The samples were analyzed in reflectance mode by a spectrophotometer FOSS NIRSystems 6500. ...

  7. Novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by PRRSV-2.

    Science.gov (United States)

    Harding, John C S; Ladinig, Andrea; Novakovic, Predrag; Detmer, Susan E; Wilkinson, Jamie M; Yang, Tianfu; Lunney, Joan K; Plastow, Graham S

    2017-09-01

    A large challenge experiment using North American porcine reproductive and respiratory virus (PRRSV-2) provided new insights into the pathophysiology of reproductive PRRS. Deep phenotyping of dams and fetuses identified maternal and fetal predictors of PRRS severity and resilience. PRRSV infection resulted in dramatic decreases in all leukocyte subsets by 2days post inoculation. Apoptosis in the interface region was positively related to endometrial vasculitis, viral load in endometrium and fetal thymus, and odds of meconium staining. Viral load at the maternal-fetal interface was a strong predictor of viral load in fetal thymus and odds of fetal death. However, interferon-alpha suppression, a consequence of PRRSV infection, was protective against fetal death. Although the prevalence of fetal lesions was low, their presence in fetal organs and umbilical cord was strongly associated with fetal compromise. Fetal death and viral load clustered in litters suggesting inter-fetal transmission starting from a limited number of index fetuses. Factors associated with index fetal infection are unclear, but large fetuses appear at greater risk. Disease progression in fetuses was associated with an up-regulation of genes associated with inflammation, innate immunity, and cell death signaling, and down-regulation of genes associated with cell cycle and lymphocyte quality. A number of maternal transcriptomic responses were associated with PRRS resilience including higher basal gene expression correlated with platelet function, interferon and pro-inflammatory responses. Twenty-one genomic regions across 10 chromosomes were associated with important traits including fetal viral load, fetal death and viability suggesting that selection for reproductive PRRS resilience may be possible. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The KnowRISK project: Tools and strategies to reduce non-structural damage

    Science.gov (United States)

    Sousa Oliveira, Carlos; Lopes, Mário; Mota de Sá, Francisco; Amaral Ferreia, Mónica; Candeias, Paulo; Campos Costa, Alfredo; Rupakhety, Rajesh; Meroni, Fabrizio; Azzaro, Raffaele; D'Amico, Salvatore; Langer, Horst; Musacchio, Gemma; Sousa Silva, Delta; Falsaperla, Susanna; Scarfì, Luciano; Tusa, Giuseppina; Tuvé, Tiziana

    2016-04-01

    The project KnowRISK (Know your city, Reduce seISmic risK through non-structural elements) is financed by the European Commission to develop prevention measures that may reduce non-structural damage in urban areas. Pilot areas of the project are within the three European participating countries, namely Portugal, Iceland and Italy. Non-structural components of a building include all those components that are not part of the structural system, more specifically the architectural, mechanical, electrical, and plumbing systems, as well as furniture, fixtures, equipment, and contents. Windows, partitions, granite veneer, piping, ceilings, air conditioning ducts and equipment, elevators, computer and hospital equipment, file cabinets, and retail merchandise are all examples of non-structural components that are vulnerable to earthquake damage. We will use the experience gained during past earthquakes, which struck in particular Iceland, Italy and Portugal (Azores). Securing the non-structural elements improves the safety during an earthquake and saves lives. This paper aims at identifying non-structural seismic protection measures in the pilot areas and to develop a portfolio of good practices for the most common and serious non-structural vulnerabilities. This systematic identification and the portfolio will be achieved through a "cross-knowledge" strategy based on previous researches, evidence of non-structural damage in past earthquakes. Shake table tests of a group of non-structural elements will be performed. These tests will be filmed and, jointly with portfolio, will serve as didactic supporting tools to be used in workshops with building construction stakeholders and in risk communication activities. A Practical Guide for non-structural risk reduction will be specifically prepared for citizens on the basis of the outputs of the project, taking into account the local culture and needs of each participating country.

  9. A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine; Hjulsager, Charlotte Kristiane; Fahnøe, Ulrik

    2013-01-01

    . In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted...... viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally....

  10. Comparison of PRRSV Nucleic Acid and Antibody Detection in Pen-Based Oral Fluid and Individual Serum Samples in Three Different Age Categories of Post-Weaning Pigs from Endemically Infected Farms.

    Directory of Open Access Journals (Sweden)

    Nick De Regge

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV is the causative agent of an economically important disease in swine. Since it has been shown that PRRSV and PRRSV specific antibodies can be detected in oral fluid, many different aspects have been studied to show that oral fluid could be a worthy alternative diagnostic sample to serum for monitoring and surveillance of this disease. Thorough field evaluations are however missing to convincingly show its usefulness under representative field conditions.Pen-based oral fluid samples and serum samples from all individual pigs in the corresponding pens were collected from post-weaning pigs of three different age categories in eight endemically PRRSV infected farms and one PRRSV free farm in Belgium. All samples were tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR and ELISA to detect PRRSV RNA and PRRSV specific antibodies, respectively.While the relative specificity of PRRSV detection by qRT-PCR in pen-based oral fluid compared to serum collected from individual pigs was high in all age categories (>90%, the relative sensitivity decreased with the age of the pigs (89, 93 and 10% in 8-12w, 16-20w and 24-28w old pigs, respectively. The latter correlated with a lower percentage of PRRSV positive pigs in serum/pen in the different age categories (55, 29 and 6%, respectively. Irrespective of the age category, pen-based oral fluid samples were always found PCR positive when at least 30% of the individual pigs were positive in serum. PRRSV specific antibody detection in oral fluid by ELISA showed a 100% relative sensitivity to detection in serum since oral fluid samples were always positive as soon as one pig in the pen was positive in serum. On the other hand, two false positive oral fluid samples in 11 pens without serum positive pigs were found, resulting in a relative specificity of 82%. Indications are however present that the oral fluid result indicated the

  11. Nonstructural urban stormwater quality measures: building a knowledge base to improve their use.

    Science.gov (United States)

    Taylor, André C; Fletcher, Tim D

    2007-05-01

    This article summarizes a research project that investigated the use, performance, cost, and evaluation of nonstructural measures to improve urban stormwater quality. A survey of urban stormwater managers from Australia, New Zealand, and the United States revealed a widespread trend of increasing use of nonstructural measures among leading stormwater management agencies, with at least 76% of 41 types of nonstructural measures being found to be increasing in use. Data gathered from the survey, an international literature review, and a multicriteria analysis highlighted four nonstructural measures of greatest potential value: mandatory town planning controls that promote the adoption of low-impact development principles and techniques; development of strategic urban stormwater management plans for a city, shire, or catchment; stormwater management measures and programs for construction/building sites; and stormwater management activities related to municipal maintenance operations such as maintenance of the stormwater drainage network and manual litter collections. Knowledge gained on the use and performance of nonstructural measures from the survey, literature review, and three trial evaluation projects was used to develop tailored monitoring and evaluation guidelines for these types of measure. These guidelines incorporate a new evaluation framework based on seven alternative styles of evaluation that range from simply monitoring whether a nonstructural measure has been fully implemented to monitoring its impact on waterway health. This research helps to build the stormwater management industry's knowledge base concerning nonstructural measures and provides a practical tool to address common impediments associated with monitoring and evaluating the performance and cost of these measures.

  12. Assimilation, partitioning, and nonstructural carbohydrates in sweet compared with grain sorghum

    International Nuclear Information System (INIS)

    Vietor, D.M.; Miller, F.R.

    1990-01-01

    Nonstructural carbohydrate concentrations in stems are greater for sweet than grain sorghums [Sorghum bicolor (L.) Moench]. Knowledge of plant characteristics associated with high nonstructural carbohydrates in sweet sorghum will air efforts to increase nonstructural carbohydrates in grain sorghum stems. This study tested the hypothesis that variation of CO 2 assimilation rate, leaf area, branching at upper nodes, and partitioning of 14 C-labeled assimilate to main stems are associated with variation of stem nonstructural carbohydrates. A sweet (Atlas X Rio) and a grain (ATx623 X RTx5388) hybrid, stages near and after physiological maturity, and defoliation and gibberellic acid (GA 3 ) treatments provided sources of variation for study. Concentrations of nonstructural carbohydrates in lower and upper stems of the sweet hybrid were 1.4 and 2.7 times higher, respectively, than for the grain hybrid, after physiological maturity. Variation in branching, including 14 C-assimilate partitioning to branches, was not consistently associated with hybrid differences in stem nonstructural carbohydrates. Increased recovery (twofold) of 14 C-assimilate in roots and labeled leaves corresponded with lower percentages of 14 C-assimilate and lower concentrations of nonstructural carbohydrates in stems of the grain hybrid. Leaf areas and leaf CO 2 exchange rate were twice as great for the sweet hybrid. Although defoliation of the sweet hybrid minimized leaf area differences between hybrids, the sweet hybrid accumulated twice as much nonstructural carbohydrates in branches after physiological maturity. Greater potentials for CO 2 assimilation and for 14 C-assimilate accumulation in mature stem tissue were associated with higher levels of stem nonstructural carbohydrates in the sweet compared with the grain hybrid

  13. Experimental inoculation of swine at various stages of gestation with a Danish isolate of porcine reproductive and respiratory syndrome virus (PRRSV)

    DEFF Research Database (Denmark)

    Kranker, Søren; Nielsen, Jens; Bille-Hansen, Vivi

    1998-01-01

    phase varied considerably, from one day to four weeks, for both darns and their offspring. Most frequently, PRRSV was isolated from lung and/or tonsil tissues from dead and euthanized piglets younger than 14 days of age. Histopathological investigations of piglets typically revealed focal nonsuppurative...

  14. Impact of PRRSV infection and dietary soybean meal on ileal amino acid digestibility and endogenous amino acid losses in growing pigs

    Science.gov (United States)

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant disease in the swine industry and increasing soybean meal (SBM) during this disease challenge may improve performance. Our objectives were to determine the impact of SBM level on apparent total tract (ATTD) and ileal (AID) ...

  15. An evaluation of interventions for reducing the risk of PRRSV introduction to filtered farms via retrograde air movement through idle fans.

    Science.gov (United States)

    Alonso, Carmen; Otake, Satoshi; Davies, Peter; Dee, Scott

    2012-06-15

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant pathogen of pigs that can be transported via the airborne route out to 9.1 km. To reduce this risk, large swine facilities have started to implement systems to filter contaminated incoming air. A proposed means of air filtration failure is the retrograde movement of air (back-drafting) from the external environment into the animal air space through non-filtered points such as idle wall fans; however, this risk has not been validated. Therefore, the purpose of this study was threefold: (1) to prove that PRRSV introduction via retrograde air movement through idle fans is a true risk; (2) to determine the minimum retrograde air velocity necessary to introduce PRRSV to an animal airspace from an external source; and (3) to evaluate the efficacy of different interventions designed to reduce this risk. A retrograde air movement model was used to test a range of velocities and interventions, including a standard plastic shutter, a plastic shutter plus a canvas cover, a nylon air chute, an aluminum shutter plus an air chute and a double shutter system. Results indicated that retrograde air movement is a real risk for PRRSV introduction to a filtered air space; however, it required a velocity of 0.76 m/s. In addition, while all the interventions designed to reduce this risk were superior when compared to a standard plastic shutter, significant differences were detected between treatments. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Use of recycled tires in non-structural concrete

    Directory of Open Access Journals (Sweden)

    Al Rawahi Zamzam

    2017-01-01

    Full Text Available This research addresses the issue of tire waste management and natural aggregate resource depletion. It investigates use of commercially produced recycled tire rubber as replacement for fine and coarse aggregate in non-structural concrete. Two replacement levels of 10% and 20% were considered for fine aggregate with 0% or 10% of coarse aggregate. The study employed a mix proportion of 1:5:4 (cement: fine aggregate: coarse aggregate with a water-to-cement ratio of 0.25, which is normally utilized in concrete block manufacturing in Oman. The mixes were tested for their thermal conductivity, water absorption and compressive strength. The behavior of mixes exposed to 100 and 200°C was also studied and the samples were later tested for compressive strength. The results showed improvements in compressive strength after exposure to heat. Thermal conductivity was reduced as the percentage replacement increased for both fine and coarse aggregate. During heat exposure, the temperature rise was faster in rubberized mixes, and the compressive strength of all mixes improved after the exposure to heat. Water absorption and void content increased with increase in replacement percentage. The compressive strength did not show a clear trend with the replacement and this is due to the sensitivity of the stiff mix used in the study and its inherent lean nature. The results indicate that the lean nature of the mix makes it insensitive to small replacement investigated in this research.

  17. Evaluation of the effectiveness of an antimicrobial air filter to avoid porcine reproductive and respiratory syndrome virus (PRRSV) aerosol transmission, after 16 months of exposure to a commercial swine environmental conditions

    Energy Technology Data Exchange (ETDEWEB)

    Batista, L. [Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO (United States); Pouliot, F.; Urizar, L. [Centre de developpement du porc du Quebec Inc., Quebec City, PQ (Canada)

    2010-07-01

    The effectiveness of Noveko's antimicrobial filter was evaluated after 16 months of exposure to commercial swine production. The experiment involved the use of a scaled model of a commercial swine facility consisting of 2 small chambers connected by a duct containing the filters. A 5 kg naive pig was placed in the reception chamber for a period of 6 hours after aerosolization with porcine reproductive and respiratory syndrome virus (PRRSV). Blood samples from pigs were collected before and after aerosolization to test for the presence of PRRSV RNA. Only blood samples were tested for PRRSV antibodies by IDEXX 2XR ELISA. None of the 9 pigs tested were found to be infected. The study showed that the technology used to integrate the antimicrobial agent into the filter fibers allows the filter combination to withstand extreme weather and endure commercial swine production for at least 16 months, and can maintain its effectiveness to avoid airborne transmission of PRRSV.

  18. Tree Nonstructural Carbohydrate Reserves Across Eastern US Temperate Forests

    Science.gov (United States)

    Mantooth, J.; Dietze, M.

    2015-12-01

    Understanding the roles, importance, and dynamics of tree non-structural carbohydrates (NSCs) is currently an active area of research. The question of how the relationships between NSCs, growth, and mortality can be used to develop more accurate projections of forest dynamics is central to this research. To begin to address this question, we have asked an even more fundamental question: How much are trees allocating carbon to storage, in the form of NSCs, versus new growth? Ecological theory predicts that there should be trade-offs between different plant life history strategies provided that there are the carbon mass-balance constraints to enforce these trade-offs. Current data on tree NSCs lack the spatial and taxonomic extent required to properly address this question. Therefore, we established a network of forest inventory plots at ten sites across the eastern US and measured growth in adult trees using increment cores and repeat measures of diameter at breast height (DBH). Increment cores were also used to measure sapwood NSCs. We hypothesized that across the eastern US, shade tolerant species, e.g. Sugar Maple (Acer saccharum) have the largest NSC reserves and that shade intolerant species have the lowest reserves. We also hypothesized that NSC reserves increase with temperature and precipitation, as with growth, and that within species NSC reserves increase with growth rate. Initial analyses of tree NSCs indicates that trees of intermediate shade tolerance, e.g. Red Oak (Quercus rubra) have the highest concentrations of sapwood NSCs, and among the highest growth rates. Across the entire study region, NSC concentrations are positively correlated with tree size and growth rate. Within species, NSC concentrations are also positively correlated with growth rate. Across functional groups healthy individuals have significantly higher sapwood NSC concentrations than visibly stressed individuals. There are also significantly lower NSC concentrations in sapwood of

  19. Fast and robust methods for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

    DEFF Research Database (Denmark)

    Kvisgaard, Lise Kirstine; Hjulsager, Charlotte Kristiane; Fahnøe, Ulrik

    . In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) of PRRSV Type 1 and Type 2 viruses were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods were shown to generate robust and reliable sequences both...... on primary material and cell culture adapted viruses and the protocols were shown to perform well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq 2000, and Ion Torrent PGM™ Sequencer). To complete the sequences at the 5’ end, 5’ Rapid Amplification of cDNA Ends (5’ RACE) was conducted...... followed by cycle sequencing of clones. The genome lengths were determined to be 14,876-15,098 and 15,342-15,408 nucleotides long for the Type 1 and Type 2 strains, respectively. These methods will greatly facilitate the generation of more complete genome PRRSV sequences globally which in turn may lead...

  20. A user exposure based approach for non-structural road network vulnerability analysis.

    Directory of Open Access Journals (Sweden)

    Lei Jin

    Full Text Available Aiming at the dense urban road network vulnerability without structural negative consequences, this paper proposes a novel non-structural road network vulnerability analysis framework. Three aspects of the framework are mainly described: (i the rationality of non-structural road network vulnerability, (ii the metrics for negative consequences accounting for variant road conditions, and (iii the introduction of a new vulnerability index based on user exposure. Based on the proposed methodology, a case study in the Sioux Falls network which was usually threatened by regular heavy snow during wintertime is detailedly discussed. The vulnerability ranking of links of Sioux Falls network with respect to heavy snow scenario is identified. As a result of non-structural consequences accompanied by conceivable degeneration of network, there are significant increases in generalized travel time costs which are measurements for "emotionally hurt" of topological road network.

  1. Seismic assessment and performance of nonstructural components affected by structural modeling

    Energy Technology Data Exchange (ETDEWEB)

    Hur, Jieun; Althoff, Eric; Sezen, Halil; Denning, Richard; Aldemir, Tunc [Ohio State University, Columbus (United States)

    2017-03-15

    Seismic probabilistic risk assessment (SPRA) requires a large number of simulations to evaluate the seismic vulnerability of structural and nonstructural components in nuclear power plants. The effect of structural modeling and analysis assumptions on dynamic analysis of 3D and simplified 2D stick models of auxiliary buildings and the attached nonstructural components is investigated. Dynamic characteristics and seismic performance of building models are also evaluated, as well as the computational accuracy of the models. The presented results provide a better understanding of the dynamic behavior and seismic performance of auxiliary buildings. The results also help to quantify the impact of uncertainties associated with modeling and analysis of simplified numerical models of structural and nonstructural components subjected to seismic shaking on the predicted seismic failure probabilities of these systems.

  2. Antibiotic-Mediated Inhibition of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV Infection: A Novel Quinolone Function Which Potentiates the Antiviral Cytokine Response in MARC-145 Cells and Pig Macrophages

    Directory of Open Access Journals (Sweden)

    William A. Cafruny

    2008-01-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV is an economically significant agent for which there currently are no effective treatments. Development of antiviral agents for PRRSV as well as many other viruses has been limited by toxicity of known antiviral compounds. In contrast, antibiotics for non-virus microbial infections have been widely useful, in part because of their acceptable toxicity in animals. We report here the discovery that the quinolonecontaining compound Plasmocin™, as well as the quinolones nalidixic acid and ciprofloxacin, have potent anti-PRRSV activity in vitro. PRRSV replication was inhibited by these antibiotics in both cultured MARC-145 cells and cultured primary alveolar porcine macrophages (PAMs. Furthermore, sub-optimal concentrations of nalidixic acid synergized with antiviral cytokines (AK-2 or IFN-γ to quantitatively and qualitatively inhibit PRRSV replication in MARC-145 cells or PAMs. The antiviral activity of Plasmocin and nalidixic acid correlated with reduced actin expression in MARC-145 cells. Replication of the related lactate dehydrogenase-elevating virus (LDV was also inhibited in primary mouse macrophages by Plasmocin. These results are significant to the development of antiviral strategies with potentially reduced toxicity, and provide a model system to better understand regulation of arterivirus replication.

  3. 33 CFR 203.50 - Nonstructural alternatives to rehabilitation of flood control works.

    Science.gov (United States)

    2010-07-01

    ... rehabilitation of flood control works. 203.50 Section 203.50 Navigation and Navigable Waters CORPS OF ENGINEERS... DISASTER PROCEDURES Rehabilitation Assistance for Flood Control Works Damaged by Flood or Coastal Storm: The Corps Rehabilitation and Inspection Program § 203.50 Nonstructural alternatives to rehabilitation...

  4. Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections

    NARCIS (Netherlands)

    P. Koraka (Penelope); C.P. Burghoorn-Maas; A. Falconar; T.E. Setiati (Tatty); K. Djamiatun; J. Groen (Jan); A.D.M.E. Osterhaus (Albert)

    2003-01-01

    textabstractAccurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot

  5. Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

    NARCIS (Netherlands)

    Koraka, P.; Burghoorn-Maas, C.P.; Falconar, A.; Setiati, T.E.; Djamiatun, K.; Groen, J.; Osterhaus, A.D.

    2003-01-01

    Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot

  6. A live-attenuated chimeric porcine circovirus type 2 (PCV2) vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) and is efficacious in a PCV2b-PRRSV-PPV challenge model.

    Science.gov (United States)

    Opriessnig, T; Shen, H G; Pal, N; Ramamoorthy, S; Huang, Y W; Lager, K M; Beach, N M; Halbur, P G; Meng, X J

    2011-08-01

    The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure.

  7. A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model▿

    Science.gov (United States)

    Opriessnig, T.; Shen, H. G.; Pal, N.; Ramamoorthy, S.; Huang, Y. W.; Lager, K. M.; Beach, N. M.; Halbur, P. G.; Meng, X. J.

    2011-01-01

    The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure. PMID:21653745

  8. Enriched Housing Reduces Disease Susceptibility to Co-Infection with Porcine Reproductive and Respiratory Virus (PRRSV) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) in Young Pigs.

    Science.gov (United States)

    van Dixhoorn, Ingrid D E; Reimert, Inonge; Middelkoop, Jenny; Bolhuis, J Elizabeth; Wisselink, Henk J; Groot Koerkamp, Peter W G; Kemp, Bas; Stockhofe-Zurwieden, Norbert

    2016-01-01

    Until today, anti-microbial drugs have been the therapy of choice to combat bacterial diseases. Resistance against antibiotics is of growing concern in man and animals. Stress, caused by demanding environmental conditions, can reduce immune protection in the host, influencing the onset and outcome of infectious diseases. Therefore psychoneuro-immunological intervention may prove to be a successful approach to diminish the impact of diseases and antibiotics use. This study was designed to investigate the effect of social and environmental enrichment on the impact of disease, referred to as "disease susceptibility", in pigs using a co-infection model of PRRSV and A. pleuropneumoniae. Twenty-eight pigs were raised in four pens under barren conditions and twenty-eight other pigs were raised in four pens under enriched conditions. In the enriched pens a combination of established social and environmental enrichment factors were introduced. Two pens of the barren (BH) and two pens of the enriched housed (EH) pigs were infected with PRRSV followed by A. pleuropneumoniae, the other two pens in each housing treatment served as control groups. We tested if differences in disease susceptibility in terms of pathological and clinical outcome were related to the different housing regimes and if this was reflected in differences in behavioural and immunological states of the animals. Enriched housed pigs showed a faster clearance of viral PRRSV RNA in blood serum (p = 0.014) and histologically 2.8 fold less interstitial pneumonia signs in the lungs (p = 0.014). More barren housed than enriched housed pigs developed lesions in the lungs (OR = 19.2, p = 0.048) and the lesions in the barren housed pigs showed a higher total pathologic tissue damage score (ppigs. EH pigs showed less stress-related behaviour and differed immunologically and clinically from BH pigs. We conclude that enriched housing management reduces disease susceptibility to co-infection of PRRSV and A

  9. Enriched Housing Reduces Disease Susceptibility to Co-Infection with Porcine Reproductive and Respiratory Virus (PRRSV and Actinobacillus pleuropneumoniae (A. pleuropneumoniae in Young Pigs.

    Directory of Open Access Journals (Sweden)

    Ingrid D E van Dixhoorn

    Full Text Available Until today, anti-microbial drugs have been the therapy of choice to combat bacterial diseases. Resistance against antibiotics is of growing concern in man and animals. Stress, caused by demanding environmental conditions, can reduce immune protection in the host, influencing the onset and outcome of infectious diseases. Therefore psychoneuro-immunological intervention may prove to be a successful approach to diminish the impact of diseases and antibiotics use. This study was designed to investigate the effect of social and environmental enrichment on the impact of disease, referred to as "disease susceptibility", in pigs using a co-infection model of PRRSV and A. pleuropneumoniae. Twenty-eight pigs were raised in four pens under barren conditions and twenty-eight other pigs were raised in four pens under enriched conditions. In the enriched pens a combination of established social and environmental enrichment factors were introduced. Two pens of the barren (BH and two pens of the enriched housed (EH pigs were infected with PRRSV followed by A. pleuropneumoniae, the other two pens in each housing treatment served as control groups. We tested if differences in disease susceptibility in terms of pathological and clinical outcome were related to the different housing regimes and if this was reflected in differences in behavioural and immunological states of the animals. Enriched housed pigs showed a faster clearance of viral PRRSV RNA in blood serum (p = 0.014 and histologically 2.8 fold less interstitial pneumonia signs in the lungs (p = 0.014. More barren housed than enriched housed pigs developed lesions in the lungs (OR = 19.2, p = 0.048 and the lesions in the barren housed pigs showed a higher total pathologic tissue damage score (p<0.001 than those in enriched housed pigs. EH pigs showed less stress-related behaviour and differed immunologically and clinically from BH pigs. We conclude that enriched housing management reduces disease

  10. Nonstructural damages of reinforced concrete buildings due to 2015 Ranau earthquake

    Science.gov (United States)

    Adiyanto, Mohd Irwan; Majid, Taksiah A.; Nazri, Fadzli Mohamed

    2017-07-01

    On 15th June 2016 a moderate earthquake with magnitude Mw5.9 was occurred in Sabah, Malaysia. Specifically, the epicentre was located at 16 km northwest of Ranau. Less than two days after the first event, a reconnaissance mission took action to investigate the damages on buildings. Since the reinforced concrete buildings in Ranau were designed based on gravity and wind load only, a lot of minor to severe damages was occurred. This paper presents the damages on the nonstructural elements of reinforced concrete buildings due to Ranau earthquake. The assessment was conducted via in-situ field investigation covering the visual observation, taking photo, and interview with local resident. Based on in-situ field investigation, there was a lot of damages occurred on the nonstructural elements like the brick walls. Such damages cannot be neglected since it can cause injury and fatality to the victims. Therefore, it can be concluded that the installation of nonstructural elements should be reviewed for the sake of safety.

  11. Different clinical, virological, serological and tissue tropism outcomes of two new and one old Belgian type 1 subtype 1 porcine reproductive and respiratory virus (PRRSV) isolates

    DEFF Research Database (Denmark)

    Frydas, Ilias S.; Trus, Ivan; Kvisgaard, Lise Kirstine

    2015-01-01

    in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils......In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed....... It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for sialoadhesin-cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication...

  12. Development of a swine specific 9-plex Luminex cytokine assay and assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination: Elevated serum IL-12 levels are not predictive of protect

    Science.gov (United States)

    A Luminex multiplex swine cytokine assay was developed to measure 9 cytokines simultaneously in pig serum and tested in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This assay detects innate (IL-1ß, IL-6, IL-8, IFNa, TNFa); regulatory (IL-10), Th1 (IL-12, I...

  13. Functional assignment to JEV proteins using SVM.

    Science.gov (United States)

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

  14. Expression of Recombinant Potato leafroll virus Structural and Non-structural Proteins for Antibody Production

    Czech Academy of Sciences Publication Activity Database

    Plchová, Helena; Moravec, Tomáš; Dědič, P.; Čeřovská, Noemi

    2011-01-01

    Roč. 159, č. 2 (2011), s. 130-132 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030; GA MZe QH71123 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato leafroll virus * recombinant viral antigen * antibody production Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.791, year: 2011

  15. Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.

    Science.gov (United States)

    Arai, Masaaki; Tsukiyama-Kohara, Kyoko; Takagi, Asako; Tobita, Yoshimi; Inoue, Kazuaki; Kohara, Michinori

    2014-05-23

    Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The KnowRISK project - Know your city, Reduce seISmic risK through non-structural elements

    Science.gov (United States)

    Sousa Oliveria, Carlos; Amaral Ferreira, Mónica; Lopez, Mário; Sousa Silva, Delta; Musacchio, Gemma; Rupakhety, Rajesh; Falsaperla, Susanna; Meroni, Fabrizio; Langer, Horst

    2016-04-01

    Historically, there is a tendency to focus on seismic structural performance of buildings, neglecting the potential for damage of non-structural elements. In particular, non-structural elements of buildings are their architectural parts (i.e. partitions, ceilings, cladding), electrical and mechanical components (i.e., distribution panels, piping, plumbing), and contents (e.g., furniture, bookcases, computers and desktop equipment). Damage of these elements often contributes significantly to earthquake impacts. In the 1999 Izmit Earthquake, Turkey, 50% of the injuries and 3% of human losses were caused by non-structural failures. In the 2010-2011 Christchurch Earthquakes (New Zealand), 40% of building damage was induced by non-structural malfunctions. Around 70%-85% of construction cost goes into these elements, and their damage can strongly influence the ability of communities to cope with and recover from earthquakes. The project Know your city, Reduce seISmic risK through non-structural elements (KnowRISK) aims at facilitating local communities' access to expert knowledge on non-structural seismic protection solutions. The project will study seismic scenarios critical for non-structural damage, produce a portfolio of non-structural protection measures and investigate the level of awareness in specific communities. We will implement risk communication strategies that will take into account the social and cultural background and a participatory approach to raise awareness in local communities. The paradox between the progress of scientific knowledge and the ongoing increase of losses from natural disasters worldwide is a well-identified gap in the UN Hyogo Framework for Action 2005-2015, in which one of the main priorities is the investment on "knowledge use, innovation and education to build a culture of safety and resilience". The KnowRISK is well aligned with these priorities and will contribute to participatory action aimed at: i) transferring expert knowledge

  17. Influence of dietary nonstructural carbohydrate concentration on growth performance and carcass characteristics of Holstein steers.

    Science.gov (United States)

    Ramos-Aviña, Daniel; Plascencia, Alejandro; Zinn, Richard

    2018-06-01

    Since very little information exists about the topic; in this experiment we compare, in a long-term finishing program, the growth-performance responses and carcass characteristics of Holstein steers where non-structural carbohydrate concentration of the diet is reduced from 64% to 51% (dry matter basis). Sixty Holstein steer calves (129±2.2 kg) were blocked by initial weight into five groups and randomly assigned within weight groupings to 10 pens. Calves were fed with a steam-flaked corn-based finishing diets containing 51% higher fiber (HF) or 64% lower fiber (LF) nonstructural carbohydrates. Non-structural carbohydrates concentrations were manipulated substituting dried distiller grain with solubles and alfalfa hay for flaked corn. Cattle were weighed every 112 days and at the end of the experiment (day 308) when the cattle were harvested and carcass characteristics were evaluated. Steers fed the HF diet showed improvement (8.8%) in average daily gain (ADG) during the initial 112-d period. This effect was followed by a numerical trend for greater ADG throughout the remainder of the study so that overall ADG tended to be greater (4.9%, p = 0.06) for the HF than for LF. There were no treatment effects on dry matter intake. Gain efficiency and estimated dietary net energy (NE) were greater 8.3% and 5.2%, respectively for HF during the initial 112-d period. Overall (308-d) gain efficiency and estimated dietary NE were similar for both dietary treatments. However, due to differences in tabular dietary NE, the ratio of observed:expected dietary NE tended to be greater (4.1%, p = 0.06) for the HF vs LF diet. There were no treatment effects on carcass characteristics except for a tendency toward a slightly greater (0.5%, p = 0.09) estimated carcass yield. Reducing the non-structural carbohydrate concentration of a conventional steam-flaked corn-based growing finishing diet for Holstein steers can effectively enhance growth performance, particularly during the early

  18. Temporary CD8(+) T-cell depletion in pigs does not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV)

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Eriksen, Lis

    2004-01-01

    Several studies have demonstrated a consistent increase in the CD8(+) T-cell subset of pigs following infection with porcine reproductive and respiratory virus (PRRSV). Consequently, it has been suggested that CD8(+) T-cells may play an important role in protection against this infection. In order...... increased disease nor influenced the ability to clear virus in the treated pigs......., confirmed the depletion effect of specific mAb therapy. Almost complete depletion of cell subsets expressing the CD8(+) antigen was obtained on day 2 and 5 post infection (PI) with nadir less than 1 % of peripheral blood mononuclear cells (PBMC). One week PI, an increase in T-cell subsets was observed...

  19. Upscaling the Use of Mixed Recycled Aggregates in Non-Structural Low Cement Concrete.

    Science.gov (United States)

    López-Uceda, Antonio; Ayuso, Jesús; Jiménez, José Ramón; Agrela, Francisco; Barbudo, Auxiliadora; De Brito, Jorge

    2016-02-02

    This research aims to produce non-structural concrete with mixed recycled aggregates (MRA) in upscaled applications with low-cement content. Four slabs were executed with concrete made with different ratios of coarse MRA (0%, 20%, 40% and 100%), using the mix design, the mixing procedures and the facilities from a nearby concrete production plant. The analysis of the long-term compressive and splitting tensile strengths in concrete cores, extracted from the slabs, allowed the highlighting of the long-term high strength development potential of MRA incorporation. The study of cast specimens produced in situ under the same conditions as the slabs showed, firstly, that the use of MRA has a great influence on the properties related to durability, secondly, that the loss of compressive strength for total MRA incorporation relative to control concrete increases proportionally with the class strength, and, thirdly, that the mechanical properties (including Schmidt hammer results) from the concrete slabs showed no significant differences relative to the control concrete for coarse aggregates replacements up to 40%. Therefore, this upscaled experimental study supports the application of concrete with 100% coarse MRA incorporation and low cement content in non-structural civil works such as bike lanes, gutters, ground slabs, leveling surfaces, and subgrades for foundations. To the best of the authors' knowledge, there have not been any upscaled applications of concrete with MRA and low cement content.

  20. Upscaling the Use of Mixed Recycled Aggregates in Non-Structural Low Cement Concrete

    Directory of Open Access Journals (Sweden)

    Antonio López-Uceda

    2016-02-01

    Full Text Available This research aims to produce non-structural concrete with mixed recycled aggregates (MRA in upscaled applications with low-cement content. Four slabs were executed with concrete made with different ratios of coarse MRA (0%, 20%, 40% and 100%, using the mix design, the mixing procedures and the facilities from a nearby concrete production plant. The analysis of the long-term compressive and splitting tensile strengths in concrete cores, extracted from the slabs, allowed the highlighting of the long-term high strength development potential of MRA incorporation. The study of cast specimens produced in situ under the same conditions as the slabs showed, firstly, that the use of MRA has a great influence on the properties related to durability, secondly, that the loss of compressive strength for total MRA incorporation relative to control concrete increases proportionally with the class strength, and, thirdly, that the mechanical properties (including Schmidt hammer results from the concrete slabs showed no significant differences relative to the control concrete for coarse aggregates replacements up to 40%. Therefore, this upscaled experimental study supports the application of concrete with 100% coarse MRA incorporation and low cement content in non-structural civil works such as bike lanes, gutters, ground slabs, leveling surfaces, and subgrades for foundations. To the best of the authors’ knowledge, there have not been any upscaled applications of concrete with MRA and low cement content.

  1. New non-structured discretizations for fluid flows with reinforced incompressibility

    International Nuclear Information System (INIS)

    Heib, S.

    2003-01-01

    This work deals with the discretization of Stokes and Navier-Stokes equations modeling the flow of incompressible fluids on 2-D or 3-D non-structured meshes. Triangles and tetrahedrons are used for 2-D and 3-D meshes, respectively. The developments and calculations are performed with the code Priceles (fast CEA-EdF industrial platform for large Eddy simulation). This code allows to perform simulations both on structured and non-structured meshes. A finite-volume resolution method is used: a finite difference volume (FDV) method is used for the structured meshes and a finite element volume (FEV) method is used for the non-structured meshes. The finite element used in the beginning of this work has several defects. Starting from this situation, the discretization is improved by adding modifications to this element and the new elements introduced are analyzed theoretically. In parallel to these analyses, the new discretizations are implemented in order to test them numerically and to confirm the theoretical analyses. The first chapter presents the physical and mathematical modeling used in this work. The second chapter treats of the discretization of Stokes equations and presents the FEV resolution method. Chapter 3 presents a first attempt of improvement of this finite element and leads to the proposal of a new element which is presented in details. The problem encountered with the new discretization leads to a modification presented in chapter 4. This new discretization gives all the expected convergence results and sometimes shows super-convergence properties. Chapter 5 deals with the study and discretization of the Navier-Stokes equations. The study of the filtered Navier-Stokes equations, used for large Eddy simulations, requires to give a particular attention to the discretization of the diffusive terms. Then, the convective terms are considered. The effects of the convective terms in the initial discretization and in the improved method are compared. The use of

  2. Water stress, shoot growth and storage of non-structural carbohydrates along a tree height gradient in a tall conifer

    Science.gov (United States)

    David R. Woodruff; Frederick C. Meinzer

    2011-01-01

    We analyzed concentrations of starch, sucrose, glucose and fructose in upper branch wood, foliage and trunk sapwood of Douglas-fir trees in height classes ranging from ~2 to ~57 m. Mean concentrations of non-structural carbohydrates (NSC) for all tissues were highest in the tallest height class and lowest in the lowest height class, and height-related trends in NSC...

  3. Detection of Immune-Complex Dissociated Nonstructural-1 (NS-1) Antigen in Patients with Acute Dengue Virus Infections

    NARCIS (Netherlands)

    P. Koraka (Penelope); C.P. Burghoorn-Maas; A. Falconar; T.E. Setiati (Tatty); K. Djamiatun; J. Groen (Jan); A.D.M.E. Osterhaus (Albert)

    2003-01-01

    textabstractAccurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot

  4. Quantitative characterization of nonstructural carbohydrates of mezcal Agave (Agave salmiana Otto ex Salm-Dick).

    Science.gov (United States)

    Michel-Cuello, Christian; Juárez-Flores, Bertha Irene; Aguirre-Rivera, Juan Rogelio; Pinos-Rodríguez, Juan Manuel

    2008-07-23

    Fructans are the reserve carbohydrates in Agave spp. plants. In mezcal factories, fructans undergoes thermal hydrolysis to release fructose and glucose, which are the basis to produce this spirit. Carbohydrate content determines the yield of the final product, which depends on plant organ, ripeness stage, and thermal hydrolysis. Thus, a qualitative and quantitative characterization of nonstructural carbohydrates was conducted in raw and hydrolyzed juices extracted from Agave salmiana stems and leaves under three ripeness stages. By high-performance liquid chromatography (HPLC), fructose, glucose, sucrose, xylose, and maltose were identified in agave juice. Only the plant fraction with hydrolysis interaction was found to be significant in the glucose concentration plant. Interactions of the fraction with hydrolysis and ripeness with hydrolysis were statistically significant in fructose concentration. Fructose concentration rose considerably with hydrolysis, but only in juice extracted from ripe agave stems (early mature and castrated). This increase was statistically significant only with acid hydrolysis.

  5. Retrieving Risk-Neutral Densities Embedded in VIX Options: a Non-Structural Approach

    DEFF Research Database (Denmark)

    Barletta, Andrea; Santucci de Magistris, Paolo; Violante, Francesco

    We propose a non-structural pricing method to retrieve the risk-neutral density implied by options contracts on the CBOE VIX. The method is based on orthogonal polynomial expansions around a kernel density and yields the risk-neutral density of the underlying asset without the need for modeling its...... dynamics. The method imposes only mild regularity conditions on shape of the density. The approach can be thought of as an alternative to Hermite expansions where the kernel has positive support. .e family of Laguerre kernels is extended to include the GIG and the generalized Weibull densities, which, due...... to their flexible rate of decay, are better suited at modeling the density of the VIX. Based on this technique, we propose a simple and robust way to estimate the expansion coefficients by means of a principal components analysis. We show that the proposed methodology yields an accurate approximation of the risk...

  6. Whole-tree distribution and temporal variation of non-structural carbohydrates in broadleaf evergreen trees.

    Science.gov (United States)

    Smith, Merryn G; Miller, Rebecca E; Arndt, Stefan K; Kasel, Sabine; Bennett, Lauren T

    2018-04-01

    Non-structural carbohydrates (NSCs) form a fundamental yet poorly quantified carbon pool in trees. Studies of NSC seasonality in forest trees have seldom measured whole-tree NSC stocks and allocation among organs, and are not representative of all tree functional types. Non-structural carbohydrate research has primarily focussed on broadleaf deciduous and coniferous evergreen trees with distinct growing seasons, while broadleaf evergreen trees remain under-studied despite their different growth phenology. We measured whole-tree NSC allocation and temporal variation in Eucalyptus obliqua L'Hér., a broadleaf evergreen tree species typically occurring in mixed-age temperate forests, which has year-round growth and the capacity to resprout after fire. Our overarching objective was to improve the empirical basis for understanding the functional importance of NSC allocation and stock changes at the tree- and organ-level in this tree functional type. Starch was the principal storage carbohydrate and was primarily stored in the stem and roots of young (14-year-old) trees rather than the lignotuber, which did not appear to be a specialized starch storage organ. Whole-tree NSC stocks were depleted during spring and summer due to significant decreases in starch mass in the roots and stem, seemingly to support root and crown growth but potentially exacerbated by water stress in summer. Seasonality of stem NSCs differed between young and mature trees, and was not synchronized with stem basal area increments in mature trees. Our results suggest that the relative magnitude of seasonal NSC stock changes could vary with tree growth stage, and that the main drivers of NSC fluctuations in broadleaf evergreen trees in temperate biomes could be periodic disturbances such as summer drought and fire, rather than growth phenology. These results have implications for understanding post-fire tree recovery via resprouting, and for incorporating NSC pools into carbon models of mixed

  7. Properties of Non-Structural Concrete Made with Mixed Recycled Aggregates and Low Cement Content.

    Science.gov (United States)

    López-Uceda, Antonio; Ayuso, Jesús; López, Martin; Jimenez, José Ramón; Agrela, Francisco; Sierra, María José

    2016-01-26

    In spite of not being legally accepted in most countries, mixed recycled aggregates (MRA) could be a suitable raw material for concrete manufacturing. The aims of this research were as follows: (i) to analyze the effect of the replacement ratio of natural coarse aggregates with MRA, the amount of ceramic particles in MRA, and the amount of cement, on the mechanical and physical properties of a non-structural concrete made with a low cement content; and (ii) to verify if it is possible to achieve a low-strength concrete that replaces a greater amount of natural aggregate with MRA and that has a low cement content. Two series of concrete mixes were manufactured using 180 and 200 kg/m³ of CEM II/A-V 42.5 R type Portland cement. Each series included seven concrete mixes: one with natural aggregates; two MRA with different ceramic particle contents; and one for each coarse aggregate replacement ratio (20%, 40%, and 100%). To study their properties, compressive and splitting tensile strength, modulus of elasticity, density, porosity, water penetration, and sorptivity, tests were performed. The results confirmed that the main factors affecting the properties analyzed in this research are the amount of cement and the replacement ratio; the two MRAs used in this work presented a similar influence on the properties. A non-structural, low-strength concrete (15 MPa) with an MRA replacement ratio of up to 100% for 200 kg/m³ of cement was obtained. This type of concrete could be applied in the construction of ditches, sidewalks, and other similar civil works.

  8. Properties of Non-Structural Concrete Made with Mixed Recycled Aggregates and Low Cement Content

    Science.gov (United States)

    López-Uceda, Antonio; Ayuso, Jesús; López, Martin; Jimenez, José Ramón; Agrela, Francisco; Sierra, María José

    2016-01-01

    In spite of not being legally accepted in most countries, mixed recycled aggregates (MRA) could be a suitable raw material for concrete manufacturing. The aims of this research were as follows: (i) to analyze the effect of the replacement ratio of natural coarse aggregates with MRA, the amount of ceramic particles in MRA, and the amount of cement, on the mechanical and physical properties of a non-structural concrete made with a low cement content; and (ii) to verify if it is possible to achieve a low-strength concrete that replaces a greater amount of natural aggregate with MRA and that has a low cement content. Two series of concrete mixes were manufactured using 180 and 200 kg/m3 of CEM II/A-V 42.5 R type Portland cement. Each series included seven concrete mixes: one with natural aggregates; two MRA with different ceramic particle contents; and one for each coarse aggregate replacement ratio (20%, 40%, and 100%). To study their properties, compressive and splitting tensile strength, modulus of elasticity, density, porosity, water penetration, and sorptivity, tests were performed. The results confirmed that the main factors affecting the properties analyzed in this research are the amount of cement and the replacement ratio; the two MRAs used in this work presented a similar influence on the properties. A non-structural, low-strength concrete (15 MPa) with an MRA replacement ratio of up to 100% for 200 kg/m3 of cement was obtained. This type of concrete could be applied in the construction of ditches, sidewalks, and other similar civil works. PMID:28787874

  9. Kobuviral Non-structural 3A Proteins Act as Molecular Harnesses to Hijack the Host ACBD3 Protein

    Czech Academy of Sciences Publication Activity Database

    Klíma, Martin; Chalupská, Dominika; Rozycki, B.; Humpolíčková, Jana; Řežábková, L.; Šilhán, Jan; Bäumlová, Adriana; Dubánková, Anna; Bouřa, Evžen

    2017-01-01

    Roč. 25, č. 2 (2017), s. 219-230 ISSN 0969-2126 R&D Projects: GA ČR(CZ) GJ17-07058Y Institutional support: RVO:61388963 Keywords : RNA replication * poliovirus 3A * Aichi virus Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.945, year: 2016 https://www.sciencedirect.com/science/article/pii/S096921261630363X?via%3Dihub

  10. Evaluation of three 3ABC ELISAs for foot-and-mouth disease non-structural antibodies using latent class analysis

    Directory of Open Access Journals (Sweden)

    Malirat Viviane

    2006-10-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious viral disease of even-toed ungulates. Serological diagnosis/surveillance of FMD presents several problems as there are seven serotypes worldwide and in the event of vaccination it may be necessary to be able to identify FMD infected/exposed animals irrespective of their vaccination status. The recent development of non-structural 3ABC protein (NSP ELISA tests has greatly advanced sero-diagnosis/surveillance as these tests detect exposure to live virus for any of the seven serotypes of FMD, even in vaccinated populations. This paper analyses the performance of three NSP tests using a Bayesian formulation of the Hui-Walter latent class model to estimate test sensitivity and specificity in the absence of a "gold-standard" test, using sera from a well described cattle population in Cameroon with endemic FMD. Results The analysis found a high sensitivity and specificity for both the Danish C-ELISA and the World Organisation for Animal Health (O.I.E. recommended South American I-ELISA. However, the commercial CHEKIT kit, though having high specificity, has very low sensitivity. The results of the study suggests that for NSP ELISAs, latent class models are a useful alternative to the traditional approach of evaluating diagnostic tests against a known "gold-standard" test as imperfections in the "gold-standard" may give biased test characteristics. Conclusion This study demonstrates that when applied to naturally infected zebu cattle managed under extensive rangeland conditions, the FMD ELISAs may not give the same parameter estimates as those generated from experimental studies. The Bayesian approach allows for full posterior probabilities and capture of the uncertainty in the estimates. The implications of an imperfect specificity are important for the design and interpretation of sero-surveillance data and may result in excessive numbers of false positives in low prevalence

  11. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

    NARCIS (Netherlands)

    Hedil, M.; Sterken, M.G.; Ronde, de D.; Lohuis, D.; Kormelink, R.

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS.

  12. Effects of non-structural components and soil-structure interaction on the seismic response of framed structures

    Science.gov (United States)

    Ditommaso, Rocco; Auletta, Gianluca; Iacovino, Chiara; Nigro, Antonella; Carlo Ponzo, Felice

    2017-04-01

    In this paper, several nonlinear numerical models of reinforced concrete framed structures have been defined in order to evaluate the effects of non-structural elements and soil-structure interaction on the elastic dynamic behaviour of buildings. In the last few years, many and various studies have highlighted the significant effects derived from the interaction between structural and non-structural components on the main dynamic characteristics of a building. Usually, structural and non-structural elements act together, adding both masses and stiffness. The presence of infill panels is generally neglected in the design process of structural elements, although these elements can significantly increase the lateral stiffness of a structure leading to a modification in the dynamic properties. Particularly, at the Damage Limit State (where an elastic behaviour is expected), soil-structure interaction effects and non-structural elements may further affect the elastic natural period of buildings, changing the spectral accelerations compared with those provided by seismic codes in case of static analyses. In this work, a parametric study has been performed in order to evaluate the elastic fundamental period of vibration of buildings as a function of structural morphology (height, plan area, ratio between plan dimensions), infills presence and distribution and soil characteristics. Acknowledgements This study was partially funded by the Italian Department of Civil Protection within the project DPC-RELUIS 2016 - RS4 ''Seismic observatory of structures and health monitoring'' and by the "Centre of Integrated Geomorphology for the Mediterranean Area - CGIAM" within the Framework Agreement with the University of Basilicata "Study, Research and Experimentation in the Field of Analysis and Monitoring of Seismic Vulnerability of Strategic and Relevant Buildings for the purposes of Civil Protection and Development of Innovative Strategies of Seismic Reinforcement".

  13. Therapeutic efficacy of pedicle screw-rod internal fixation after one-stage posterior transforaminal lesion debridement and non-structural bone grafting for tuberculosis of lumbar vertebra

    Directory of Open Access Journals (Sweden)

    Jia-ming LIU

    2015-11-01

    Full Text Available Objective To evaluate the efficacy and safety of pedicle screw-rod internal fixation after one-stage posterior transforaminal lesion debridement and non-structural bone grafting in the treatment of tuberculosis of mono-segmental lumbar vertebra. Methods From January 2010 to April 2013, 21 patients (9 males and 12 females with an average age of 49.1 years with mono-segmental tuberculosis of lumbar vertebra underwent surgery in our hospital were included. Eight patients had neurological deficit. The focus of tuberculosis was located on one side of the vertebral body, and all the patients had obvious signs of bone destruction on CT and MRI. All the patients were given anti-tuberculosis chemotherapy for 2-3 weeks before surgery. The local bone chips and autologous iliac cancellous bone were used as the intervertebral bone graft. Postoperative plain radiographs and CT were obtained to evaluate the fusion rate and degree of lumbar lordosis. The visual analogue scale score (VAS, erythrocyte sedimentation rate (ESR, and C-reactive protein (CRP before and after operation, and at final follow-up date were recorded. Results All the patients were followed up for 25.3±4.2 months. The mean operation time was 157±39 minutes, and the average blood loss was 470±143ml. The fusion rate of the interbody bone graft was 95.2%, with an average fusion period of 6.1±2.5 months. The neurological function was improved by 100%, and no severe complication or neurological injury occured. The preoperative and postoperative lordosis angles of the lumbar spine were 21.4°±5.7° and 33.6°±3.1°, respectively, and it was 31.3°±2.7° at the final follow up. The preoperative and postoperative VAS scores were 7.8±2.6 and 2.4±1.7 respectively, and it was 0.9±0.7 at the final follow up. The ESR and CRP were significantly decreased 3 months after surgery, and they became normal at 6 months. Conclusion Pedicle screw-rod internal fixation after one-stage posterior

  14. Distribution and mixing of old and new nonstructural carbon in two temperate trees.

    Science.gov (United States)

    Richardson, Andrew D; Carbone, Mariah S; Huggett, Brett A; Furze, Morgan E; Czimczik, Claudia I; Walker, Jennifer C; Xu, Xiaomei; Schaberg, Paul G; Murakami, Paula

    2015-04-01

    We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two temperate trees, and we used the continuous label offered by the radiocarbon (carbon-14, (14) C) bomb spike to estimate the mean age of NSC in different tissues. NSC in branches and the outermost stemwood growth rings had the (14) C signature of the current growing season. However, NSC in older aboveground and belowground tissues was enriched in (14) C, indicating that it was produced from older assimilates. Radial patterns of (14) C in stemwood NSC showed strong mixing of NSC across the youngest growth rings, with limited 'mixing in' of younger NSC to older rings. Sugars in the outermost five growth rings, accounting for two-thirds of the stemwood pool, had a mean age  5 yr. Our results are thus consistent with a previously-hypothesized two-pool ('fast' and 'slow' cycling NSC) model structure. These pools appear to be physically distinct. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  15. Improving Rice Modeling Success Rate with Ternary Non-structural Fertilizer Response Model.

    Science.gov (United States)

    Li, Juan; Zhang, Mingqing; Chen, Fang; Yao, Baoquan

    2018-06-13

    Fertilizer response modelling is an important technical approach to realize metrological fertilization on rice. With the goal of solving the problems of a low success rate of a ternary quadratic polynomial model (TPFM) and to expand the model's applicability, this paper established a ternary non-structural fertilizer response model (TNFM) based on the experimental results from N, P and K fertilized rice fields. Our research results showed that the TNFM significantly improved the modelling success rate by addressing problems arising from setting the bias and multicollinearity in a TPFM. The results from 88 rice field trials in China indicated that the proportion of typical TNFMs that satisfy the general fertilizer response law of plant nutrition was 40.9%, while the analogous proportion of TPFMs was only 26.1%. The recommended fertilization showed a significant positive linear correlation between the two models, and the parameters N 0 , P 0 and K 0 that estimated the value of soil supplying nutrient equivalents can be used as better indicators of yield potential in plots where no N or P or K fertilizer was applied. The theoretical analysis showed that the new model has a higher fitting accuracy and a wider application range.

  16. Nonstructural carbohydrate dynamics of lodgepole pine dying from mountain pine beetle attack.

    Science.gov (United States)

    Wiley, Erin; Rogers, Bruce J; Hodgkinson, Robert; Landhäusser, Simon M

    2016-01-01

    Bark beetle outbreaks are an important cause of tree death, but the process by which trees die remains poorly understood. The effect of beetle attack on whole-tree nonstructural carbohydrate (NSC) dynamics is particularly unclear, despite the potential role of carbohydrates in plant defense and survival. We monitored NSC dynamics of all organs in attacked and protected lodgepole pines (Pinus contorta) during a mountain pine beetle (Dendroctonus ponderosae) outbreak in British Columbia, starting before beetle flight in June 2011 through October 2012, when most attacked trees had died. Following attack, NSC concentrations were first reduced in the attacked region of the bole. The first NSC reduction in a distant organ appeared in the needles at the end of 2011, while branch and root NSC did not decline until much later in 2012. Attacked trees that were still alive in October 2012 had less beetle damage, which was negatively correlated with initial bark sugar concentrations in the attack region. The NSC dynamics of dying trees indicate that trees were killed by a loss of water conduction and not girdling. Further, our results identify locally reduced carbohydrate availability as an important mechanism by which stressors like drought may increase tree susceptibility to biotic attack. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  17. Drought stress, growth and nonstructural carbohydrate dynamics of pine trees in a semi-arid forest.

    Science.gov (United States)

    Klein, Tamir; Hoch, Günter; Yakir, Dan; Körner, Christian

    2014-09-01

    In trees exposed to prolonged drought, both carbon uptake (C source) and growth (C sink) typically decrease. This correlation raises two important questions: (i) to what degree is tree growth limited by C availability; and (ii) is growth limited by concurrent C storage (e.g., as nonstructural carbohydrates, NSC)? To test the relationships between drought, growth and C reserves, we monitored the changes in NSC levels and constructed stem growth chronologies of mature Pinus halepensis Miller trees of three drought stress levels growing in Yatir forest, Israel, at the dry distribution limit of forests. Moderately stressed and stressed trees showed 34 and 14% of the stem growth, 71 and 31% of the sap flux density, and 79 and 66% of the final needle length of healthy trees in 2012. In spite of these large reductions in growth and sap flow, both starch and soluble sugar concentrations in the branches of these trees were similar in all trees throughout the dry season (2-4% dry mass). At the same time, the root starch concentrations of moderately stressed and stressed trees were 47 and 58% of those of healthy trees, but never drought there is more than one way for a tree to maintain a positive C balance. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Age, allocation and availability of nonstructural carbon in mature red maple trees.

    Science.gov (United States)

    Carbone, Mariah S; Czimczik, Claudia I; Keenan, Trevor F; Murakami, Paula F; Pederson, Neil; Schaberg, Paul G; Xu, Xiaomei; Richardson, Andrew D

    2013-12-01

    The allocation of nonstructural carbon (NSC) to growth, metabolism and storage remains poorly understood, but is critical for the prediction of stress tolerance and mortality. We used the radiocarbon ((14) C) 'bomb spike' as a tracer of substrate and age of carbon in stemwood NSC, CO2 emitted by stems, tree ring cellulose and stump sprouts regenerated following harvesting in mature red maple trees. We addressed the following questions: which factors influence the age of stemwood NSC?; to what extent is stored vs new NSC used for metabolism and growth?; and, is older, stored NSC available for use? The mean age of extracted stemwood NSC was 10 yr. More vigorous trees had both larger and younger stemwood NSC pools. NSC used to support metabolism (stem CO2 ) was 1-2 yr old in spring before leaves emerged, but reflected current-year photosynthetic products in late summer. The tree ring cellulose (14) C age was 0.9 yr older than direct ring counts. Stump sprouts were formed from NSC up to 17 yr old. Thus, younger NSC is preferentially used for growth and day-to-day metabolic demands. More recently stored NSC contributes to annual ring growth and metabolism in the dormant season, yet decade-old and older NSC is accessible for regrowth. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  19. The buffering capacity of stems: genetic architecture of nonstructural carbohydrates in cultivated Asian rice, Oryza sativa.

    Science.gov (United States)

    Wang, Diane R; Han, Rongkui; Wolfrum, Edward J; McCouch, Susan R

    2017-07-01

    Harnessing stem carbohydrate dynamics in grasses offers an opportunity to help meet future demands for plant-based food, fiber and fuel production, but requires a greater understanding of the genetic controls that govern the synthesis, interconversion and transport of such energy reserves. We map out a blueprint of the genetic architecture of rice (Oryza sativa) stem nonstructural carbohydrates (NSC) at two critical developmental time-points using a subpopulation-specific genome-wide association approach on two diverse germplasm panels followed by quantitative trait loci (QTL) mapping in a biparental population. Overall, 26 QTL are identified; three are detected in multiple panels and are associated with starch-at-maturity, sucrose-at-maturity and NSC-at-heading. They tag OsHXK6 (rice hexokinase), ISA2 (rice isoamylase) and a tandem array of sugar transporters. This study provides the foundation for more in-depth molecular investigation to validate candidate genes underlying rice stem NSC and informs future comparative studies in other agronomically vital grass species. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  20. The Sustainable Island Development Evaluation Model and Its Application Based on the Nonstructural Decision Fuzzy Set

    Directory of Open Access Journals (Sweden)

    Quanming Wang

    2013-01-01

    Full Text Available Due to the complexity and diversity of the issue of sustainable island development, no widely accepted and applicable evaluation system model regarding the issue currently exists. In this paper, we discuss and establish the sustainable development indicator system and the model approach from the perspective of resources, the island environment, the island development status, the island social development, and the island intelligence development. We reference the sustainable development theory and the sustainable development indicator system method concerning land region, combine the character of the sustainable island development, analyze and evaluate the extent of the sustainable island development, orient development, and identify the key and limited factors of sustainable island development capability. This research adopts the entropy method and the nonstructural decision fuzzy set theory model to determine the weight of the evaluating indicators. Changhai County was selected as the subject of the research, which consisted of a quantitative study of its sustainable development status from 2001 to 2008 to identify the key factors influencing its sustainability development, existing problems, and limited factors and to provide basic technical support for ocean development planning and economic development planning.

  1. Preparation for emergence of an Eastern European porcine reproductive and respiratory syndrome virus (PRRSV) strain in Western Europe: Immunization with modified live virus vaccines or a field strain confers partial protection.

    Science.gov (United States)

    Renson, P; Fablet, C; Le Dimna, M; Mahé, S; Touzain, F; Blanchard, Y; Paboeuf, F; Rose, N; Bourry, O

    2017-05-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses for the swine industry worldwide. In the past several years, highly pathogenic strains that lead to even greater losses have emerged. For the Western European swine industry, one threat is the possible introduction of Eastern European PRRSV strains (example Lena genotype 1.3) which were shown to be more virulent than common Western resident strains under experimental conditions. To prepare for the possible emergence of this strain in Western Europe, we immunized piglets with a Western European PRRSV field strain (Finistere: Fini, genotype 1.1), a new genotype 1 commercial modified live virus (MLV) vaccine (MLV1) or a genotype 2 commercial MLV vaccine (MLV2) to evaluate and compare the level of protection that these strains conferred upon challenge with the Lena strain 4 weeks later. Results show that immunization with Fini, MLV1 or MLV2 strains shortened the Lena-induced hyperthermia. In the Fini group, a positive effect was also demonstrated in growth performance. The level of Lena viremia was reduced for all immunized groups (significantly so for Fini and MLV2). This reduction in Lena viremia was correlated with the level of Lena-specific IFNγ-secreting cells. In conclusion, we showed that a commercial MLV vaccine of genotype 1 or 2, as well as a field strain of genotype 1.1 may provide partial clinical and virological protection upon challenge with the Lena strain. The cross-protection induced by these immunizing strains was not related with the level of genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is attributed to a better cell-mediated immune response. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication

    International Nuclear Information System (INIS)

    Lee, Changhee; Hodgins, Douglas; Calvert, Jay G.; Welch, Siao-Kun W.; Jolie, Rika; Yoo, Dongwan

    2006-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a

  3. Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

    Directory of Open Access Journals (Sweden)

    Luping Du

    2018-01-01

    Full Text Available Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide (PLGA nanoparticles (NPs with Ulex europaeus agglutinin 1 (UEA-1 and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5] or subunit vaccine ORF5-encoded glycoprotein (GP5 from exposure to the gastrointestinal (GI tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05. Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system.

  4. Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

    Science.gov (United States)

    Du, Luping; Yu, Zhengyu; Pang, Fengjiao; Xu, Xiangwei; Mao, Aihua; Yuan, Wanzhe; He, Kongwang; Li, Bin

    2018-01-01

    Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells) within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with Ulex europaeus agglutinin 1 (UEA-1) and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5)] or subunit vaccine ORF5-encoded glycoprotein (GP5) from exposure to the gastrointestinal (GI) tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05). Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system. PMID:29423381

  5. Can gas exchange dynamics predict non-structural carbohydrate use under drought stress?

    Science.gov (United States)

    Kannenberg, S.; Phillips, R.

    2016-12-01

    A recent conceptual framework for understanding tree drought responses characterizes species along a continuum from isohydry to anisohydry, with theory predicting that isohydric and anisohydric trees should display different carbon (C) allocation patterns under drought conditions. We tested the hypothesis that the trade-offs inherent in the isohydry-anisohydry framework (i.e., C starvation vs. hydraulic failure) necessitate different allocation patterns to non-structural carbohydrates (NSCs), growth, and respiration. Specifically, we hypothesized that isohydric trees would decrease NSC stores and growth in the face of reduced incoming photoassimilate, whereas anisohydric trees would maintain assimilation, growth, and NSC pools due to decreased demand for stored metabolic C and enhanced osmoregulatory needs. To test this, we subjected saplings of Liriodendron tulipifera (an isohydric tree) and Quercus alba (an anisohydric tree) to a six week drought in the greenhouse, and measured assimilation, leaf water potential (midday and predawn), growth, leaf dark respiration and NSCs (both sugars and starch in aboveground and belowground tissues) in control and droughted plants. Overall, we confirmed that the isohydric and anisohydric species used NSCs differently during drought. In most tissues, both species had similar responses of NSCs to drought: starch NSCs were maintained or decreased while sugar NSCs tended to increase. Stem NSCs were a notable exception, as L. tulipifera decreased total NSC to almost zero while NSCs in Q. alba remained constant. This depletion of stem NSC in L. tulipifera was offset by increases in other tissues, however, resulting in no net change to total NSC during the drought. In contrast, Q. alba increased total NSC. Interestingly, Q. alba also decreased assimilation and growth, indicating a potential trade-off between NSC and biomass allocation. Our results show that NSCs in different tissues may have contrasting uses as storage or

  6. Reoperation for non-structural valvular dysfunction caused by pannus ingrowth in aortic valve prosthesis.

    Science.gov (United States)

    Oh, Se Jin; Park, Samina; Kim, Jun Sung; Kim, Kyung-Hwan; Kim, Ki Bong; Ahn, Hyuk

    2013-07-01

    The authors' clinical experience is presented of non-structural valvular dysfunction of the prosthetic aortic valve caused by pannus ingrowth during the late postoperative period after previous heart valve surgery. Between January 1999 and April 2012, at the authors' institution, a total of 33 patients underwent reoperation for increased mean pressure gradient of the prosthetic aortic valve. All patients were shown to have pannus ingrowth. The mean interval from the previous operation was 16.7 +/- 4.3 years, and the most common etiology for the previous aortic valve replacement (AVR) was rheumatic valve disease. The mean effective orifice area index (EOAI) of the previous prosthetic valve was 0.97 +/- 0.11 cm2/m2, and the mean pressure gradient on the aortic prosthesis before reoperation was 39.1 +/- 10.7 mmHg. Two patients (6.1%) died in-hospital, and late death occurred in six patients (18.2%). At the first operation, 30 patients underwent mitral or tricuspid valve surgery as a concomitant procedure. Among these operations, mitral valve replacement (MVR) was combined in 24 of all 26 patients with rheumatic valve disease. Four patients underwent pannus removal only while the prosthetic aortic valve was left in place. The mean EOAI after reoperation was significantly increased to 1.16 +/- 0.16 cm2/m2 (p pannus ingrowth was shown in patients with a small EOAI of the prosthetic aortic valve and combined MVR for rheumatic disease. As reoperation for pannus overgrowth showed good clinical outcomes, an aggressive resection of pannus and repeated AVR should be considered in symptomatic patients to avoid the complications of other cardiac diseases.

  7. Spectroscopic Remote Sensing of Non-Structural Carbohydrates in Forest Canopies

    Directory of Open Access Journals (Sweden)

    Gregory P. Asner

    2015-03-01

    Full Text Available Non-structural carbohydrates (NSC are products of photosynthesis, and leaf NSC concentration may be a prognostic indicator of climate-change tolerance in woody plants. However, measurement of leaf NSC is prohibitively labor intensive, especially in tropical forests, where foliage is difficult to access and where NSC concentrations vary enormously by species and across environments. Imaging spectroscopy may allow quantitative mapping of leaf NSC, but this possibility remains unproven. We tested the accuracy of NSC remote sensing at leaf, canopy and stand levels using visible-to-shortwave infrared (VSWIR spectroscopy with partial least squares regression (PLSR techniques. Leaf-level analyses demonstrated the high precision (R2 = 0.69–0.73 and accuracy (%RMSE = 13%–14% of NSC estimates in 6136 live samples taken from 4222 forest canopy species worldwide. The leaf spectral data were combined with a radiative transfer model to simulate the role of canopy structural variability, which led to a reduction in the precision and accuracy of leaf NSC estimation (R2 = 0.56; %RMSE = 16%. Application of the approach to 79 one-hectare plots in Amazonia using the Carnegie Airborne Observatory VSWIR spectrometer indicated the good precision and accuracy of leaf NSC estimates at the forest stand level (R2 = 0.49; %RMSE = 9.1%. Spectral analyses indicated strong contributions of the shortwave-IR (1300–2500 nm region to leaf NSC determination at all scales. We conclude that leaf NSC can be remotely sensed, opening doors to monitoring forest canopy physiological responses to environmental stress and climate change.

  8. Efficacy of double-stranded RNA against white spot syndrome virus (WSSV non-structural (orf89, wsv191 and structural (vp28, vp26 genes in the Pacific white shrimp Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    César M. Escobedo-Bonilla

    2015-04-01

    Full Text Available White spot syndrome virus (WSSV is a major pathogen in shrimp aquaculture. RNA interference (RNAi is a promising tool against viral infections. Previous works with RNAi showed different antiviral efficacies depending on the silenced gene. This work evaluated the antiviral efficacy of double-stranded (ds RNA against two non-structural (orf89, wsv191 WSSV genes compared to structural (vp26, vp28 genes to inhibit an experimental WSSV infection. Gene orf89 encodes a putative regulatory protein and gene white spot virus (wsv191 encodes a nonspecific nuclease; whereas genes vp26 and vp28 encode envelope proteins, respectively. Molecules of dsRNA against each of the WSSV genes were intramuscularly injected (4 μg per shrimp into a group of shrimp 48 h before a WSSV challenge. The highest antiviral activity occurred with dsRNA against orf89, vp28 and vp26 (cumulative mortalities 10%, 10% and 21%, respectively. In contrast, the least effective treatment was wsv191 dsRNA (cumulative mortality 83%. All dead animals were WSSV-positive by one-step PCR, whereas reverse-transcription PCR of all surviving shrimp confirmed inhibition of virus replication. This study showed that dsRNA against WSSV genes orf89, vp28 and vp26 were highly effective to inhibit virus replication and suggest an essential role in WSSV infection. Non-structural WSSV genes such as orf89 can be used as novel targets to design therapeutic RNAi molecules against WSSV infection.

  9. Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Costello, F.; Huhtanen, M.

    1996-01-01

    Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP...

  10. NIRS determination of non-structural carbohydrates, water soluble carbohydrates and other nutritive quality traits in whole plant maize with wide range variability

    Directory of Open Access Journals (Sweden)

    L. Campo

    2013-05-01

    Full Text Available The aim of this work was to study the potential of near-infrared reflectance spectroscopy (NIRS to predict non-structural carbohydrates (NSC, water soluble carbohydrates (WSC, in vitro organic dry matter digestibility (IVOMD, organic matter (OM, crude protein (CP, neutral detergent fiber (NDF, acid detergent fiber (ADF and starch in samples of whole plant maize with a wide range of variability. The samples were analyzed in reflectance mode by a spectrophotometer FOSS NIRSystems 6500. Four hundred and fifty samples of wide spectrum from different origin were selected out of 3000 scanned for the calibration set, whereas 87 independent random samples were used in the external validation. The goodness of the calibration models was evaluated using the following statistics: coefficient of determination (R2, standard error of cross-validation (SECV, standard error of prediction for external validation (SEP and the RPDCV and RPDP indexes [ratios of standard deviation (SD of reference analysis data to SECV and SEP, respectively]. The smaller the SECV and SEP and the greater the RPDCV and RPDP, the predictions are better. Trait measurement units were g/100g of dry matter (DM, except for IVOMD (g/100g OM. The SECV and RPDCV statistics of the calibration set were 1.34 and 3.2 for WSC, 2.57 and 3 for NSC and 2.3 and 2.2 for IVOMD, respectively. The SEP and RPDP statistics for external validation were 0.74 and 4.7 for WSC, 2.14 and 2.5 for NSC and 1.68 and 1.6 for IVOMD respectively. It can be concluded that the NIRS technique can be used to predict WSC and NSC with good accuracy, whereas prediction of IVOMD showed a lesser accuracy. NIRS predictions of OM, CP, NDF, ADF and starch also showed good accuracy.

  11. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

    Science.gov (United States)

    Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus...

  12. Mutations in the Nonstructural Protein 3A Confer Resistance to the Novel Enterovirus Replication Inhibitor TTP-8307

    NARCIS (Netherlands)

    de Palma, Armando M.; Thibaut, Hendrik Jan; van der Linden, Lonneke; Lanke, Kjerstin; Heggermont, Ward; Ireland, Stephen; Andrews, Robert; Arimilli, Murty; Al-tel, Taleb H.; de Clercq, Erik; van Kuppeveld, Frank; Neyts, Johan

    2009-01-01

    A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all

  13. Alphavirus-based Vaccines Encoding Nonstructural Proteins of Hepatitis C Virus Induce Robust and Protective T-cell Responses

    NARCIS (Netherlands)

    Ip, Peng; Boerma, Annemarie; Regts, Joke; Meijerhof, Tjarko; Wilschut, Jan; Nijman, Hans W.; Daemen, Toos

    An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all-or a part of

  14. Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection

    NARCIS (Netherlands)

    Diepolder, H. M.; Gerlach, J. T.; Zachoval, R.; Hoffmann, R. M.; Jung, M. C.; Wierenga, E. A.; Scholz, S.; Santantonio, T.; Houghton, M.; Southwood, S.; Sette, A.; Pape, G. R.

    1997-01-01

    In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient's immune system and the viral population seem to be crucial in

  15. Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus.

    Science.gov (United States)

    Bhatt, Mukesh; Mohapatra, Jajati K; Pandey, Laxmi K; Mohanty, Nihar N; Das, Biswajit; Prusty, Bikash R; Pattnaik, Bramhadev

    2018-01-02

    Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87-144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A 87-144 3B 1,2 +FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log 10 7.42 TCID 50 /ml) and LFBK-α V β 6 (log 10 8.30 TCID 50 /ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A 87-153 3B 1,2 I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control. Copyright © 2017 Elsevier B.V. All rights

  16. Tula hantavirus NSs protein accumulates in the perinuclear area in infected and transfected cells.

    Science.gov (United States)

    Virtanen, Jussi Oskari; Jääskeläinen, Kirsi Maria; Djupsjöbacka, Janica; Vaheri, Antti; Plyusnin, Alexander

    2010-01-01

    The small RNA segment of some hantaviruses (family Bunyaviridae) encodes two proteins: the nucleocapsid protein and, in an overlapping reading frame, a non-structural (NSs) protein. The hantavirus NSs protein, like those of orthobunya- and phleboviruses, counteracts host innate immunity. Here, for the first time, the NSs protein of a hantavirus (Tula virus) has been observed in infected cells and shown to localize in the perinuclear area. Transiently expressed NSs protein showed similar localization, although the kinetics was slightly different, suggesting that to reach its proper location in the infected cell, the NSs protein does not have to cooperate with other viral proteins.

  17. Comparison of commercial and experimental porcine circovirus type 2 (PCV2) vaccines using a triple challenge with PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV).

    Science.gov (United States)

    Shen, H G; Beach, N M; Huang, Y W; Halbur, P G; Meng, X J; Opriessnig, T

    2010-08-23

    The efficacies of commercial porcine circovirus type 2 (PCV2) vaccines and a live PCV1-2a chimeric vaccine were compared in conventional, PCV2-positive piglets using a PCV2-porcine reproductive and respiratory syndrome virus (PRRSV)-porcine parvovirus (PPV) coinfection challenge model. Seventy-three, 2-week-old pigs were randomized into seven groups including five vaccinated and two control groups. Pigs in the vaccinated groups were vaccinated at 3 weeks (one dose) or at 3 and 6 weeks (two dose) of age. All vaccine regimens tested were effective in reducing naturally occurring PCV2 viremia at 16 weeks of age and after PCV2 challenge, demonstrating the capability of the products to induce a lasting protective immunity despite the presence of PCV2 viremia at the time of vaccination. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Effects of structured versus non-structured learning on achievement and attitudes of fifth graders in a public aquarium

    Science.gov (United States)

    Kafka, Merryl Audrey

    The investigator analyzed the main effect of a structured-learning experience in an informal setting, as well as interactions between the students' learning-style variations toward the element of structure and the imposed instructional conditions. The subjects consisted of 170 students enrolled in two public schools located in Brooklyn, New York. The students were predominantly a White multi-ethnic population consisting of 118 Caucasians, 25 Hispanics, 24 Asians, and 3 African-Americans. Three randomly assigned classes (n = 81) were provided trip sheets, which directed students on how to learn new information with written questions and directives. Three randomly assigned non-structured classes (n = 89) experienced the same exhibit in a free-form manner. Science-based criterion-referenced pre- and posttests were administered, in addition to Learning Style Inventories (Dunn, Dunn, & Price, 1996) and a modified Semantic Differential Scale (Pizzo, 1981), which was used to measure attitudinal levels. The non-structured group had access to similar content information in the form of exhibit graphics, but apparently they chose not to read it as carefully or engage in the information-seeking process as intensely as the students equipped with trip sheets. Analysis of covariance (ANCOVA) indicated that a structured-learning experience produced significantly higher science-achievement test scores than in a non-structured-learning experience (p = .0001). In addition, there was no single learning-style variation (preference, aversion, or no preference) to structure that produced significantly higher gains than another. Furthermore, attitudinal scores were not significantly different between structured and non-structured groups, as well as among homogeneous subsets of students with learning-style variations that matched, mismatched, or indicated no-preferenced positions on the element of structure. Hence, a moderate amount of structure resulted in academic gains without

  19. Low Non-structured Antiretroviral Therapy Interruptions in HIV-Infected Persons Who Inject Drugs Receiving Multidisciplinary Comprehensive HIV Care at an Outpatient Drug Abuse Treatment Center.

    Science.gov (United States)

    Vallecillo, Gabriel; Mojal, Sergio; Roquer, Albert; Samos, Pilar; Luque, Sonia; Martinez, Diana; Martires, Paula Karen; Torrens, Marta

    2016-05-01

    Continuous HIV treatment is necessary to ensure successful combined antiretroviral therapy (cART). The aim of this study was to evaluate the incidence of patient-initiated non-structured treatment interruptions in HIV-infected persons who inject drugs and who received a multidisciplinary comprehensive program, including medical HIV care, drug-dependence treatment and psychosocial support, at a drug outpatient addiction center. Non-structured treatment interruptions were defined as ≥30 consecutive days off cART without medical indication. During a median follow-up of 53.8 months, 37/132 (28 %) patients experienced the first non-structured treatment interruptions. The cumulative probability of cART interruption at 5 years was 31.2 % (95 % CI 22.4-40.0). Current drug use injection ≥1/day (HR 14.77; 95 % CI 5.90-36.96) and cART naive patients (HR 0.35, 95 % CI 0.14-0.93) were predictive factors for non-structured treatment interruptions. HIV care provided at a drug addiction center is a useful strategy to sustain continuous cART, however, drug abstinence is essential for the long-term maintenance of cART.

  20. Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

    Science.gov (United States)

    Lu, Zen H; Brown, Alexander; Wilson, Alison D; Calvert, Jay G; Balasch, Monica; Fuentes-Utrilla, Pablo; Loecherbach, Julia; Turner, Frances; Talbot, Richard; Archibald, Alan L; Ait-Ali, Tahar

    2014-03-04

    Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

  1. [Non-structural abnormalities of CNS function resulting in coincidence of endocrinopathies, epilepsy and psychoneurologic disorders in children and adolescents].

    Science.gov (United States)

    Starzyk, Jerzy; Pituch-Noworolska, Anna; Pietrzyk, Jacek A; Urbanik, Andrzej; Kroczka, Sławomir; Drozdz, Ryszard; Wójcik, Małgorzata

    2010-01-01

    In the population of children and adolescents, epilepsy affects approximately 1% of cases, nonepileptic seizures are seen in approximately 3%, and endocrine disorders are several times more common. For this reason, coincidence of endocrine disorders and epilepsy and psychoneurologic disorders is frequent. Much less common are structural abnormalities (tumors, developmental abnormalities), and especially non-structural CNS abnormalities, resulting in coincidence of both disorders. There are no reports available in the literature that would address the problem. 1) Assessment of the frequency of coincidental epilepsy and endocrine disorders in patients without structural CSN abnormalities treated as outpatients and inpatients of Department of Endocrinology University Children's Hospital of Krakow. 2) Presentation of diagnostic and therapeutic difficulties in these patients, and 3) An attempt at defining the common etiology of both disorders. On the basis of ICD code patients with coincidance of endocrine disorders, epilepsy and psychoneurologic disorders were selected from several thousands of children treated between 2000 and 2009 in Pediatric Endocrinology Department. The neurologic disorders were diagnosed and treated in Chair and Department of Children's and Adolescents Neurology or in another pediatric neurology center. Various forms of epilepsy (symptomatic or idiopathic) and other psychoneurological disorders (disorders of behavior and emotions, obsession-compulsion syndromes, stereotypias, aggression, autoaggression, or hypothalamic obesity) coincident with one or more endocrine disorders, such as growth disorders, disorders of pubertal development, obesity, thyroid diseases, adrenal diseases, hyperprolactinemia, hypoparathyroidism and ion metabolism disorders were diagnosed in 49 patients. The group included: i) children after cranial irradiation and chemotherapy due to medulloblastoma (3 patients), oligodenroglioma (1 patient), ependymoma (1 patient), optic

  2. A Comparison of Effectiveness of Structured and Non-Structured Strategies of Rhetorical Invention for Written Argumentation Produced by Community College Students

    OpenAIRE

    Smolova, Alona A

    1999-01-01

    A recent shift in the composition studies has resulted in the renewal of interest in rhetorical invention. There is no uniformity among researchers and professionals about the optimal conditions preceding the composing process, especially among college students. This study was intended to explore the effectiveness of structured (Larson's Heuristic) and non-structured (freewriting) strategies of rhetorical invention produced by community college students. The objectives of this study were to d...

  3. Intra-annual dynamics of non-structural carbohydrates in the cambium of mature conifer trees reflects radial growth demands.

    Science.gov (United States)

    Simard, Sonia; Giovannelli, Alessio; Treydte, Kerstin; Traversi, Maria Laura; King, Gregory M; Frank, David; Fonti, Patrick

    2013-09-01

    The presence of soluble carbohydrates in the cambial zone, either from sugars recently produced during photosynthesis or from starch remobilized from storage organs, is necessary for radial tree growth. However, considerable uncertainties on carbohydrate dynamics and the consequences on tree productivity exist. This study aims to better understand the variation in different carbon pools at intra-annual resolution by quantifying how cambial zone sugar and starch concentrations fluctuate over the season and in relation to cambial phenology. A comparison between two physiologically different species growing at the same site, i.e., the evergreen Picea abies Karst. and the deciduous Larix decidua Mill., and between L. decidua from two contrasting elevations, is presented to identify mechanisms of growth limitation. Results indicate that the annual cycle of sugar concentration within the cambial zone is coupled to the process of wood formation. The highest sugar concentration is observed when the number of cells in secondary wall formation and lignification stages is at a maximum, subsequent to most radial growth. Starch disappears in winter, while other freeze-resistant non-structural carbohydrates (NSCs) increase. Slight differences in NSC concentration between species are consistent with the differing climate sensitivity of the evergreen and deciduous species investigated. The general absence of differences between elevations suggests that the cambial activity of trees growing at the treeline was not limited by the availability of carbohydrates at the cambial zone but instead by environmental controls on the growing season duration.

  4. Non-Structural Carbohydrate Dynamics in Leaves and Branches of Pinus massoniana (Lamb. Following 3-Year Rainfall Exclusion

    Directory of Open Access Journals (Sweden)

    Tian Lin

    2018-06-01

    Full Text Available Drought-induced tree mortality is an increasing and global ecological problem. Stored non-structural carbohydrates (NSCs may be a key determinant of drought resistance, but most existing studies are temporally limited. In this study, a 3-year 100% rainfall exclusion manipulation experiment was conducted to evaluate the response of NSC dynamics to drought stress in 25-year-old Pinus massoniana leaves and branches. The results showed: (1 compared with the control condition, leaf NSC concentration in the drought treatment increased 90% in the early stage (days 115–542 (p < 0.05, and then decreased 15% in the late stage (days 542–1032, which was attributed to water limitation instead of phenology; (2 the response of leaf NSCs to drought was more significant than branch NSCs, demonstrating a time lag effect; and (3 the response of P. massoniana to mild drought stress was to increase the soluble sugars and starch in the early stage, followed by an increase in soluble sugars caused by decreasing starch in the later stress period. Considering these results, mid-term drought stress had no significant effect on the total NSC concentration in P. massoniana, removing carbon storage as a potential adaptation to drought stress.

  5. Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

    Science.gov (United States)

    Babst, Benjamin A; Karve, Abhijit A; Judt, Tatjana

    2013-06-01

    Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

  6. The amount of parenchyma and living fibers affects storage of nonstructural carbohydrates in young stems and roots of temperate trees.

    Science.gov (United States)

    Plavcová, Lenka; Hoch, Günter; Morris, Hugh; Ghiasi, Sara; Jansen, Steven

    2016-04-01

    Concentrations of nonstructural carbohydrates (NSCs) are used as proxies for the net carbon balance of trees and as indicators of carbon starvation resulting from environmental stress. Woody organs are the largest NSC-storing compartments in forest ecosystems; therefore, it is essential to understand the factors that affect the size of this important storage pool. In wood, NSC are predominantly deposited in ray and axial parenchyma (RAP); however, direct links between nutrient storage and RAP anatomy have not yet been established. Here, we tested whether the NSC storage capacity of wood is influenced by the amount of RAP. We measured NSC concentrations and RAP fractions in root and stem sapwood of 12 temperate species sampled at the onset of winter dormancy and in stem sapwood of four tropical trees growing in an evergreen lowland rainforest. The patterns of starch distribution were visualized by staining with Lugol's solution. The concentration of NSCs in sapwood of temperate trees scales tightly with the amount of RAP and living fibers (LFs), with almost all RAP and LFs being densely packed with starch grains. In contrast, the tropical species had lower NSC concentrations despite their higher RAP and LFs fraction and had considerable interspecific differences in starch distribution. The differences in RAP and LFs abundance affect the ability of sapwood to store NSC in temperate trees, whereas a more diverse set of functions of RAP might be pronounced in species growing in a tropical environment with little seasonality. © 2016 Botanical Society of America.

  7. TOTAL NON-STRUCTURAL CARBOHYDRATE (TNC OF THREE CULTIVARS OF NAPIER GRASS (Pennisetum purpureum AT VEGETATIVE AND REPRODUCTIVE PHASE

    Directory of Open Access Journals (Sweden)

    B. Budiman

    2014-10-01

    Full Text Available An experiment was conducted to determine Total Non-structural carbohydrates (TNC of threecultivars of napier grass (Pennisetum purpureum harvested at vegetative and reproductive phases. Thecultivars tested were Taiwan (Gt, King (Gk and Mott (Gm and arranged in a 3 x 2 of treatments withfour replicates following nested design. The results showed that the highest sugar content (P<0.01 wasfound in Gt cultivar and the lowest was in Gm cultivar. The highest starch content (P<0.01 was found inGk cultivar and the lowest was in Gt cultivar. TNC content of Gt and Gk cultivars were not significantlydifferent, but both were significantly higher (P<0.01 compared with the Gm cultivar. It can beconcluded, that there were differences in TNC between cultivars, however, the TNC content in Gkcultivar was not different with Gt cultivar, while Gm cultivar have the lowest (P<0.01 TNC content. Atreproductive phase all cultivars have higher (P<0.01 TNC and starch content than at vegetative phase

  8. Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal for avirulence and RNA silencing suppression

    NARCIS (Netherlands)

    Ronde, de D.; Pasquier, A.; Ying, S.; Butterbach, P.B.E.; Lohuis, D.; Kormelink, R.J.M.

    2014-01-01

    Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS)

  9. Dynamics of non-structural carbohydrates in three Mediterranean woody species following long-term experimental drought.

    Science.gov (United States)

    Rosas, Teresa; Galiano, Lucía; Ogaya, Romà; Peñuelas, Josep; Martínez-Vilalta, Jordi

    2013-01-01

    Stored non-structural carbohydrates (NSC) have been proposed as a key determinant of drought resistance in plants. However, the evidence for this role is controversial, as it comes mostly from observational, short-term studies. Here, we take advantage of a long-term experimental throughfall reduction to elucidate the response of NSC to increased drought 14 years after the beginning of the treatment in three Mediterranean resprouter trees (Quercus ilex L., Arbutus unedo L. and Phillyrea latifolia L.). In addition, we selected 20 Q. ilex individuals outside the experimental plots to directly assess the relationship between defoliation and NSC at the individual level. We measured the seasonal course of NSC concentrations in leaves, branches and lignotuber in late winter, late spring, summer, and autumn 2012. Total concentrations of NSC were highest in the lignotuber for all species. In the long-term drought experiment we found significant depletion in concentrations of total NSC in treatment plots only in the lignotuber of A. unedo. At the same time, A. unedo was the only species showing a significant reduction in BAI under the drought treatment during the 14 years of the experiment. By contrast, Q. ilex just reduced stem growth only during the first 4 years of treatment and P. latifolia remained unaffected over the whole study period. However, we found a clear association between the concentrations of NSC and defoliation in Q. ilex individuals sampled outside the experimental plots, with lower total concentrations of NSC and lower proportion of starch in defoliated individuals. Taken together, our results suggest that stabilizing processes, probably at the stand level, may have been operating in the long-term to mitigate any impact of drought on NSC levels, and highlight the necessity to incorporate long-term experimental studies of plant responses to drought.

  10. Dynamics of non-structural carbohydrates in three Mediterranean woody species following long-term experimental drought

    Directory of Open Access Journals (Sweden)

    Teresa eRosas

    2013-10-01

    Full Text Available Stored non-structural carbohydrates (NSC have been proposed as a key determinant of drought resistance in plants. However, the evidence for this role is controversial, as it comes mostly from observational, short-term studies. Here, we take advantage of a long-term experimental throughfall reduction to elucidate the response of NSC to increased drought 14 years after the beginning of the treatment in three Mediterranean woody species (Quercus ilex L., Arbutus unedo L. and Phillyrea latifolia L.. In addition, we selected 20 Q. ilex individuals outside the experimental plots to directly assess the relationship between defoliation and NSC at the individual level. We measured the seasonal course of NSC concentrations in leaves, branches and lignotuber in late winter, late spring, summer and autumn 2012. Total concentrations of NSC were highest in the lignotuber for all species. In the long-term drought experiment we found significant depletion in concentrations of total NSC in treatment plots only in the lignotuber of A. unedo. At the same time, A. unedo was the only species showing a significant reduction in BAI under the drought treatment during the 14 years of the experiment. By contrast, Q. ilex just reduced stem growth only during the first 4 years of treatment and P. latifolia remained unaffected over the whole study period. However, we found a clear association between the concentrations of NSC and defoliation in Q. ilex individuals sampled outside the experimental plots, with lower total concentrations of NSC and lower proportion of starch in defoliated individuals. Taken together, our results suggest that stabilizing processes, probably at the stand level, may have been operating in the long-term to mitigate any impact of drought on NSC levels, and highlight the necessity to incorporate long-term experimental studies of plant responses to drought.

  11. Impacts of leaf age and heat stress duration on photosynthetic gas exchange and foliar nonstructural carbohydrates in Coffea arabica.

    Science.gov (United States)

    Marias, Danielle E; Meinzer, Frederick C; Still, Christopher

    2017-02-01

    Given future climate predictions of increased temperature, and frequency and intensity of heat waves in the tropics, suitable habitat to grow ecologically, economically, and socially valuable Coffea arabica is severely threatened. We investigated how leaf age and heat stress duration impact recovery from heat stress in C. arabica . Treated plants were heated in a growth chamber at 49°C for 45 or 90 min. Physiological recovery was monitored in situ using gas exchange, chlorophyll fluorescence (the ratio of variable to maximum fluorescence, F V / F M ), and leaf nonstructural carbohydrate (NSC) on mature and expanding leaves before and 2, 15, 25, and 50 days after treatment. Regardless of leaf age, the 90-min treatment resulted in greater F V / F M reduction 2 days after treatment and slower recovery than the 45-min treatment. In both treatments, photosynthesis of expanding leaves recovered more slowly than in mature leaves. Stomatal conductance ( g s ) decreased in expanding leaves but did not change in mature leaves. These responses led to reduced intrinsic water-use efficiency with increasing heat stress duration in both age classes. Based on a leaf energy balance model, aftereffects of heat stress would be exacerbated by increases in leaf temperature at low g s under full sunlight where C. arabica is often grown, but also under partial sunlight. Starch and total NSC content of the 45-min group significantly decreased 2 days after treatment and then accumulated 15 and 25 days after treatment coinciding with recovery of photosynthesis and F V / F M . In contrast, sucrose of the 90-min group accumulated at day 2 suggesting that phloem transport was inhibited. Both treatment group responses contrasted with control plant total NSC and starch, which declined with time associated with subsequent flower and fruit production. No treated plants produced flowers or fruits, suggesting that short duration heat stress can lead to crop failure.

  12. Feedbacks between earlywood anatomy and non-structural carbohydrates affect spring phenology and wood production in ring-porous oaks

    Science.gov (United States)

    Pérez-de-Lis, Gonzalo; García-González, Ignacio; Rozas, Vicente; Olano, José Miguel

    2016-10-01

    Non-structural carbohydrates (NSC) play a central role in the construction and maintenance of a tree's vascular system, but feedbacks between the NSC status of trees and wood formation are not fully understood. We aimed to evaluate multiple dependencies among wood anatomy, winter NSC, and phenology for coexisting temperate (Quercus robur) and sub-Mediterranean (Q. pyrenaica) oaks along a water-availability gradient in the NW Iberian Peninsula. Sapwood NSC concentrations were quantified at three sites in December 2012 (N = 240). Leaf phenology and wood anatomy were surveyed in 2013. Structural equation modelling was used to analyse the interplay among hydraulic diameter (Dh), winter NSC, budburst date, and earlywood vessel production (EVP), while the effect of Dh and EVP on latewood width was assessed by using a mixed-effects model. NSC and wood production increased under drier conditions for both species. Q. robur showed a narrower Dh and lower soluble sugar (SS) concentration (3.88-5.08 % dry matter) than Q. pyrenaica (4.06-5.57 % dry matter), but Q. robur exhibited larger EVP and wider latewood (1403 µm) than Q. pyrenaica (667 µm). Stem diameter and Dh had a positive effect on SS concentrations, which were related to an earlier leaf flushing in both species. Sapwood sugar content appeared to limit EVP exclusively in Q. pyrenaica. In turn, Dh and EVP were found to be key predictors of latewood growth. Our results confirm that sapwood SS concentrations are involved in modulating growth resumption and xylem production in spring. Q. pyrenaica exhibited a tighter control of carbohydrate allocation to wood formation than Q. robur, which would play a role in protecting against environmental stress in the sub-Mediterranean area.

  13. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

    OpenAIRE

    Hedil, Marcio; Sterken, Mark G.; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silen...

  14. FLOOD RESILIENCE AND SUSTAINABLE DEVELOPMENT IN URBAN NIGERIA: INTEGRATING TRADITIONAL AND NON-STRUCTURAL METHODS OF MITIGATING AND ADAPTING TO FLOODING IN CROSS RIVER STATE, SOUTH-EASTERN NIGERIA (II

    Directory of Open Access Journals (Sweden)

    RICHARD INGWE

    2013-04-01

    Full Text Available Flood resilience and sustainable development in urban Nigeria: integrating traditional and non-structural methods of mitigating and adapting to flooding in cross river state, south-eastern Nigeria. We examined application of non-structural measures in addition to conventional structural approaches by Government Agency and community for flood management in Cross River State (Nigeria at: regional-ambit and community levels. We used focus group discussion in depth interview, and observation methods to collect data from primary and secondary sources. Our findings include: emphasis on structural flood control measures by government agencies contrasted to use of rudimentary non-structural approaches by communities. Conceptual frames proposed for managing disasters include: emphasizing future climate change impacts based on multiple scales (temporal, spatial and societal and emphasizing historical response to disasters without increasing the visibility of climate change. We conclude that community institutions, non-government/civil society organizations should lead public institutions in promoting flood resilience based on integrated non-structural to structural measures and show recent developments regarding civil society coalition committed towards promoting environmental governance in Nigeria. Frequent flooding associated with huge losses of lives and property in the study areas, as in most of urban Nigeria, persuade us to recommend that strategically placed civil society be supported by donor/funding organizations to promote integrated non-structural and traditional-structural measures to achieve urban flood resilience nationwide.

  15. FLOOD RESILIENCE AND SUSTAINABLE DEVELOPMENT IN URBAN NIGERIA: INTEGRATING TRADITIONAL AND NON-STRUCTURAL METHODS OF MITIGATING AND ADAPTING TO FLOODING IN CROSS RIVER STATE, SOUTH-EASTERN NIGERIA (I

    Directory of Open Access Journals (Sweden)

    RICHARD INGWE

    2012-12-01

    Full Text Available Flood resilience and sustainable development in urban Nigeria: integrating traditional and non-structural methods of mitigating and adapting to flooding in cross river state, south-eastern Nigeria. We examined application of non-structural measures in addition to conventional structural approaches by Government Agency and community for flood management in Cross River State (Nigeria at: regional-ambit and community levels. We used focus group discussion in depth interview, and observation methods to collect datafrom primary and secondary sources. Our findings include: emphasis on structural flood control measures by government agencies contrasted to use of rudimentary non-structural approaches by communities. Conceptual frames proposed for managing disasters include: emphasizing future climate change impacts based on multiple scales (temporal, spatial and societal and emphasizing historical response to disasters without increasing the visibility of climate change. We conclude that community institutions, non-government/civil society organizations should lead public institutions in promoting flood resilience based on integrated non-structural to structural measures and show recent developments regarding civil society coalition committed towards promoting environmental governance in Nigeria. Frequent flooding associated with huge losses of lives and property in the studyareas, as in most of urban Nigeria, persuade us to recommend that strategically placed civil society be supported by donor/funding organizations to promote integrated non-structural and traditional-structural measures to achieve urban flood resilience nationwide.

  16. Development of NIR calibration models to assess year-to-year variation in total non-structural carbohydrates in grasses using PLSR

    DEFF Research Database (Denmark)

    Shetty, Nisha; Gislum, René; Jensen, Anne Mette Dahl

    2012-01-01

    Near-infrared (NIR) spectroscopy was used in combination with chemometrics to quantify total nonstructural carbohydrates (TNC) in grass samples in order to overcome year-to-year variation. A total of 1103 above-ground plant and root samples were collected from different field and pot experiments...... and with various experimental designs in the period from 2001 to 2005. A calibration model was developed using partial least squares regression (PLSR). The calibration model on a large data set spanning five years demonstrated that quantification of TNC using NIR spectroscopy was possible with an acceptable low...

  17. Evaluation of glucose and insulin response to haylage diets with different content of nonstructural carbohydrates in 2 breeds of horses.

    Science.gov (United States)

    Lindåse, S; Müller, C; Nostell, K; Bröjer, J

    2018-04-09

    Information about the effect of nonstructural carbohydrates (NSCs) in forage on the postprandial glucose and insulin response in horses is scarce. This is of interest as postprandial hyperinsulinemia in horses is a risk factor for laminitis. In addition, insulin sensitivity (IS) differs between breeds. The aim was to evaluate the postprandial glucose and insulin response to haylage diets with different NSC content in horses of 2 different breeds and to evaluate the relationship between the postprandial insulin response and measures of IS derived from a frequently sampled intravenous glucose tolerance test (FSIGTT). Standardbreds (n = 9) and Icelandic horses (n = 9) with a mean body condition score of 5.5 ± 0.6 (scale 1-9) were studied. Horses were clinically healthy at the start of the study and had no history of endocrinopathic laminitis. The experiment was conducted as a replicate 3 × 3 Latin square, in which horses were fed haylage diets with low (4.2%), medium (13.6%), and high (18.2%) NSC content of dry matter. Blood sampling was performed before feeding and every 30 min until 300 min after feeding. An FSIGTT was also performed in all horses. The early (first 60 min) and the total (300 min) postprandial glucose and insulin response (area under the curve [AUC]) was higher after a meal of both medium and high NSC haylage in comparison with low NSC haylage when both breeds were combined (P ≤ 0.02). There was a main effect of breed for the early (P ≤ 0.004) but not for the total (P > 0.12) postprandial glucose and insulin response. The IS index was comparable between breeds (P = 0.75). The natural logarithm of the peak concentration, the AUC for the first 60 min and the total AUC for insulin, after a meal of medium and high NSC haylage, were moderately negatively correlated (P haylage with low NSC content (P > 0.054). This study demonstrates that the postprandial insulin response is affected by both the NSC content of haylage and the horse's IS. However

  18. Osmolality and non-structural carbohydrate composition in the secondary phloem of trees across a latitudinal gradient in Europe

    Directory of Open Access Journals (Sweden)

    Anna eLintunen

    2016-06-01

    Full Text Available Phloem osmolality and its components are involved in basic cell metabolism, cell growth, and in various physiological processes including the ability of living cells to withstand drought and frost. Osmolality and sugar composition responses to environmental stresses have been extensively studied for leaves, but less for the secondary phloem of plant stems and branches. Leaf osmotic concentration and the share of pinitol and raffinose among soluble sugars increase with increasing drought or cold stress, and osmotic concentration is adjusted with osmoregulation. We hypothesize that similar responses occur in the secondary phloem of branches. We collected living bark samples from branches of adult Pinus sylvestris, Picea abies, Betula pendula and Populus tremula trees across Europe, from boreal Northern Finland to Mediterranean Portugal. In all studied species, the observed variation in phloem osmolality was mainly driven by variation in phloem water content, while tissue solute content was rather constant across regions. Osmoregulation, in which osmolality is controlled by variable tissue solute content, was stronger for Betula and Populus in comparison to the evergreen conifers. Osmolality was lowest in mid-latitude region, and from there increased by 37% towards northern Europe and 38% towards southern Europe due to low phloem water content in these regions. The ratio of raffinose to all soluble sugars was negligible at mid-latitudes and increased towards north and south, reflecting its role in cold and drought tolerance. For pinitol, another sugar known for contributing to stress tolerance, no such latitudinal pattern was observed. The proportion of sucrose was remarkably low and that of hexoses (i.e. glucose and fructose high at mid-latitudes. The ratio of starch to all non-structural carbohydrates increased towards the northern latitudes in agreement with the build-up of osmotically inactive C reservoir that can be converted into soluble

  19. Effect of regulated deficit irrigation on the morphology, physiology, carbon allocation and nonstructural carbohydrates of three Kentucky bluegrasses

    International Nuclear Information System (INIS)

    Liu, J. R.; Ma, L.; Liu, Y. K.; Liu, T. J.; Lu, J. N.; Wang, D. N.

    2015-01-01

    Regulated deficit irrigation (RDI) has been assessed in a wide number of field and fruit crops. However, few are the studies dealing with turfgrass. This study was conducted to investigate the morphology, physiology and carbon metabolic responses to regulated deficit irrigation for three Kentucky bluegrass (Poa pratensis L.) cultivars. Three Kentucky bluegrass cultivars were grown in PVC (polyvinyl chloride) tubes in a greenhouse and subjected to three soil water treatments in a growth chamber: 1) full irrigation; 2) drought stress, 21 days without water after full irrigation; and 3) drought recovery, stressed plants were re-watered for an additional 21 d. The present study indicated that drought resulted in a decline in turf quality (TQ), leaf relative water content (RWC), and photochemical efficiency (Fv/Fm) and an increase in electrolyte leakage (EL) for the cultivars. The turf quality, RWC, and Fv/Fm of the three Kentucky bluegrass cultivars increased with re-watering. The allocation of /sup 14/ C increased in the roots of these cultivars during the initial phase of drought stress, where a /sup 14/ C distribution shift from the roots to the stem and leaves appeared with further drought stress. Moreover, there was a significant accumulation of total nonstructural carbohydrates (TNC) in the leaves and stem. The TNC content in the leaves, stem, and roots did not completely return to the control levels following 21 d of re-watering, which was consistent with the recovery of TQ, RWC, Fv/Fm, and EL. In addition, during the re-watering treatment, the reduction in the TNC content may be due to increases in the demand or usage as a result of a rapid recovery in the growth and physiological activities as shown by increased TQ, RWC, and Fv/Fm and decreased EL. Our results suggested that the changes in the carbon allocation model and the accumulation and storage of TNC, as well as the changes in TQ, RWC, Fv/Fm, and EL, for the three cultivars are an adaptive reaction to

  20. Single-provenance mature conifers show higher non-structural carbohydrate storage and reduced growth in a drier location.

    Science.gov (United States)

    Piper, Frida I; Fajardo, Alex; Hoch, Günter

    2017-08-01

    Since growth is more sensitive to drought than photosynthesis, trees inhabiting dry regions are expected to exhibit higher carbohydrate storage and less growth than their conspecifics from more humid regions. However, the same pattern can be the result of different genotypes inhabiting contrasting humidity conditions. To test if reduced growth and high carbohydrate storage are environmentally driven by drought, we examined the growth and non-structural carbohydrate (NSC) concentrations in single-provenance stands of mature trees of Pinus contorta Douglas and Pinus ponderosa Douglas ex C. Lawson planted at contrasting humidity conditions (900 versus 300 mm of annual precipitation) in Patagonia, Chile. Individual tree growth was measured for each species and at each location as mean basal area increment of the last 10 years (BAI10), annual shoot elongation for the period 2011-14, and needle length for 2013 and 2014 cohorts. Additionally, needle, branch, stem sapwood and roots were collected from each sampled tree to determine soluble sugars, starch and total NSC concentrations. The two species showed lower mean BAI10 and 2013 needle length in the dry site; P. ponderosa also had lower annual shoot extension for 2011 and 2014, and lower 2014 needle length, in the dry than in the mesic site. By contrast, NSC concentrations of all woody tissues for both species were either similar or higher in the dry site when compared with the mesic site. Patterns of starch and sugars were substantially different: starch concentrations were similar between sites except for roots of P. ponderosa, which were higher in the dry site, while sugar concentrations of all woody tissues in both species were higher in the dry site. Overall, our study provides evidence that reduced growth along with carbon (C) accumulation is an environmentally driven response to drought. Furthermore, the significant accumulation of low-molecular weight sugars in the dry site is compatible with a prioritized C

  1. The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

    Science.gov (United States)

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-02-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

  2. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    Science.gov (United States)

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema

    International Nuclear Information System (INIS)

    Sandrin, Carla Zuliani; Figueiredo-Ribeiro, Rita de Cassia Leone; Carvalho, Maria Angela Machado de; Carvalho Delitti, Welington Braz; Domingos, Marisa

    2008-01-01

    This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in Sao Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment. - Particulate matter and air temperature increased non-structural carbohydrates in the standardized biomonitor grass in Sao Paulo

  4. Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema

    Energy Technology Data Exchange (ETDEWEB)

    Sandrin, Carla Zuliani; Figueiredo-Ribeiro, Rita de Cassia Leone; Carvalho, Maria Angela Machado de [Instituto de Botanica, Caixa Postal 3005, 01061-970 Sao Paulo, SP (Brazil); Carvalho Delitti, Welington Braz [Instituto de Biociencias, Universidade de Sao Paulo, Departamento de Ecologia, Caixa Postal 11461, 05422-970 Sao Paulo, SP (Brazil); Domingos, Marisa [Instituto de Botanica, Caixa Postal 3005, 01061-970 Sao Paulo, SP (Brazil)], E-mail: mmingos@superig.com.br

    2008-12-15

    This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in Sao Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment. - Particulate matter and air temperature increased non-structural carbohydrates in the standardized biomonitor grass in Sao Paulo.

  5. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

    Directory of Open Access Journals (Sweden)

    Ji'an Pan

    Full Text Available Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12 provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

  6. Physical qualification and improvements of the numerical model of a method of characteristics for the resolution of the neutron transport equation in non-structured grids

    International Nuclear Information System (INIS)

    Santandrea, Simone

    2001-01-01

    This research thesis addresses the resolution of the neutron transport equation inside reactor cells in non-structured grids and in general geometry by using the method of characteristics (MoC) and two acceleration methods developed during this research. The author introduces the MoC with a flat approximation of the neutron collision source within each computation area. This formulation leads to a linear approximation. The next part presents the mathematical framework for the use of the Lanczos iterative scheme. A new acceleration method is then introduced. The last part reports realistic cases with a high spatial and angular heterogeneity. Results obtained by using the Apollo2-TDT code are compared with those obtained with the Tripoli4 Monte-Carlo code [fr

  7. Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton, via the non-structural protein 3Cpro

    DEFF Research Database (Denmark)

    Armer, Hannah; Moffat, Katy; Wileman, Thomas

    2008-01-01

    Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus r....... In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect -tubulin distribution, and the microtubule network remained relatively unaffected....

  8. Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors

    DEFF Research Database (Denmark)

    Ramirez, Santseharay; Li, Yi-Ping; Jensen, Sanne B

    2014-01-01

    UNLABELLED: Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only ...

  9. Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle.

    NARCIS (Netherlands)

    Paton, D.J.; Clerq, De K.; Greiner, M.; Dekker, A.; Brocchi, E.; Bergmann, I.E.; Sammin, D.J.; Gubbins, S.; Parida, S.

    2006-01-01

    There has been much debate about the use of the so-called ¿vaccinate-to-live¿ policy for the control of foot-and-mouth disease (FMD) in Europe, according to which, spread of the FMD virus (FMDV) from future outbreaks could be controlled by a short period of ¿emergency¿ vaccination of surrounding

  10. Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle

    DEFF Research Database (Denmark)

    Paton, D.J.; de Clercq, K.; Greiner, Matthias

    2006-01-01

    There has been much debate about the use of the so-called "vaccinate-to-live" policy for the control of foot-and-mouth disease (FMD) in Europe, according to which, spread of the FMD virus (FMDV) from future outbreaks could be controlled by a short period of "emergency" vaccination of surrounding...... herds, reducing the need for large-scale pre-emptive culling of at-risk animals. Since vaccinated animals may become subclinically infected with FMDV following challenge exposure, it is necessary to either remove all vaccinates (vaccinate-to-kill) or to detect and remove vaccinates in which virus......) of FMDV, which are induced by infection with the virus, but not by vaccination with purified FMD vaccines. Using test sensitivity and specificity data established at a recent workshop on NSP assays [Brocchi E, Bergmann I, Dekker A, Paton DJ, Sammin DJ, Greiner M, et al. Comparative performance of six...

  11. Rift Valley fever virus NSs protein functions and the similarity to other bunyavirus NSs proteins.

    Science.gov (United States)

    Ly, Hoai J; Ikegami, Tetsuro

    2016-07-02

    Rift Valley fever is a mosquito-borne zoonotic disease that affects both ruminants and humans. The nonstructural (NS) protein, which is a major virulence factor for Rift Valley fever virus (RVFV), is encoded on the S-segment. Through the cullin 1-Skp1-Fbox E3 ligase complex, the NSs protein promotes the degradation of at least two host proteins, the TFIIH p62 and the PKR proteins. NSs protein bridges the Fbox protein with subsequent substrates, and facilitates the transfer of ubiquitin. The SAP30-YY1 complex also bridges the NSs protein with chromatin DNA, affecting cohesion and segregation of chromatin DNA as well as the activation of interferon-β promoter. The presence of NSs filaments in the nucleus induces DNA damage responses and causes cell-cycle arrest, p53 activation, and apoptosis. Despite the fact that NSs proteins have poor amino acid similarity among bunyaviruses, the strategy utilized to hijack host cells are similar. This review will provide and summarize an update of recent findings pertaining to the biological functions of the NSs protein of RVFV as well as the differences from those of other bunyaviruses.

  12. Leaf non-structural carbohydrate allocation and C:N:P stoichiometry in response to light acclimation in seedlings of two subtropical shade-tolerant tree species.

    Science.gov (United States)

    Xie, Hongtao; Yu, Mukui; Cheng, Xiangrong

    2018-03-01

    Light availability greatly affects plant growth and development. In shaded environments, plants must respond to reduced light intensity to ensure a regular rate of photosynthesis to maintain the dynamic balance of nutrients, such as leaf non-structural carbohydrates (NSCs), carbon (C), nitrogen (N) and phosphorus (P). To improve our understanding of the nutrient utilization strategies of understory shade-tolerant plants, we compared the variations in leaf NSCs, C, N and P in response to heterogeneous controlled light conditions between two subtropical evergreen broadleaf shade-tolerant species, Elaeocarpus sylvestris (E. sylvestris) and Illicium henryi (I. henryi). Light intensity treatments were applied at five levels (100%, 52%, 33%, 15% and 6% full sunlight) for 30 weeks to identify the effects of reduced light intensity on leaf NSC allocation patterns and leaf C:N:P stoichiometry characteristics. We found that leaf soluble sugar, starch and NSC concentrations in E. sylvestris showed decreasing trends with reduced light intensity, whereas I. henryi presented slightly increasing trends from 100% to 15% full sunlight and then significant decreases at extremely low light intensity (6% full sunlight). The soluble sugar/starch ratio of E. sylvestris decreased with decreasing light intensity, whereas that of I. henryi remained stable. Moreover, both species exhibited increasing trends in leaf N and P concentrations but limited leaf N:P and C:P ratio fluctuations with decreasing light intensity, revealing their adaptive strategies for poor light environments and their growth strategies under ideal light environments. There were highly significant correlations between leaf NSC variables and C:N:P stoichiometric variables in both species, revealing a trade-off in photosynthesis production between leaf NSC and carbon allocation. Thus, shade-tolerant plants readjusted their allocation of leaf NSCs, C, N and P in response to light acclimation. Redundancy analysis showed

  13. Sequence Analysis and Phylogenetic Profiling of the Nonstructural (NS Genes of H9N2 Influenza A Viruses Isolated in Iran during 1998-2007

    Directory of Open Access Journals (Sweden)

    Ebrahimi, M.

    2014-11-01

    Full Text Available The earliest evidences on circulation of Avian Influenza (AI virus on the Iranian poultry farms date back to 1998. Great economic losses through dramatic drop in egg production and high mortality rates are characteristically attributed to H9N2 AI virus. In the present work non-structural (NS genes of 10 Iranian H9N2 chicken AI viruses collected during 1998-2007 were fully sequenced and subjected to a phylogenetic analysis. The observations proved allele A was the single-detectable type of the NS gene within the studied isolates. All the examined Iranian isolates fell into the Korean sublineage with a relatively broad sequence homology (91.6-98% in nucleotide construction of the NS genes. The motif for PDZ ligand recognition of the group one isolates was either EDEV (N=6 or ESEV (N=1 While all viruses as group two contained a PL motif “KSEV” (N=3. The present work provides useful epidemiological data at molecular level on source and contemporary evolution of H9N2 virus population in Iran.

  14. Dynamic allocation and transfer of non-structural carbohydrates, a possible mechanism for the explosive growth of Moso bamboo (Phyllostachys heterocycla)

    Science.gov (United States)

    Song, Xinzhang; Peng, Changhui; Zhou, Guomo; Gu, Honghao; Li, Quan; Zhang, Chao

    2016-01-01

    Moso bamboo can rapidly complete its growth in both height and diameter within only 35–40 days after shoot emergence. However, the underlying mechanism for this “explosive growth” remains poorly understood. We investigated the dynamics of non-structural carbohydrates (NSCs) in shoots and attached mature bamboos over a 20-month period. The results showed that Moso bamboos rapidly completed their height and diameter growth within 38 days. At the same time, attached mature bamboos transferred almost all the NSCs of their leaves, branches, and especially trunks and rhizomes to the “explosively growing” shoots via underground rhizomes for the structural growth and metabolism of shoots. Approximately 4 months after shoot emergence, this transfer stopped when the leaves of the young bamboos could independently provide enough photoassimilates to meet the carbon demands of the young bamboos. During this period, the NSC content of the leaves, branches, trunks and rhizomes of mature bamboos declined by 1.5, 23, 28 and 5 fold, respectively. The trunk contributed the most NSCs to the shoots. Our findings provide new insight and a possible rational mechanism explaining the “explosive growth” of Moso bamboo and shed new light on understanding the role of NSCs in the rapid growth of Moso bamboo. PMID:27181522

  15. Nonstructural leaf carbohydrate dynamics of Pinus edulis during drought-induced tree mortality reveal role for carbon metabolism in mortality mechanism.

    Science.gov (United States)

    Adams, Henry D; Germino, Matthew J; Breshears, David D; Barron-Gafford, Greg A; Guardiola-Claramonte, Maite; Zou, Chris B; Huxman, Travis E

    2013-03-01

    Vegetation change is expected with global climate change, potentially altering ecosystem function and climate feedbacks. However, causes of plant mortality, which are central to vegetation change, are understudied, and physiological mechanisms remain unclear, particularly the roles of carbon metabolism and xylem function. We report analysis of foliar nonstructural carbohydrates (NSCs) and associated physiology from a previous experiment where earlier drought-induced mortality of Pinus edulis at elevated temperatures was associated with greater cumulative respiration. Here, we predicted faster NSC decline for warmed trees than for ambient-temperature trees. Foliar NSC in droughted trees declined by 30% through mortality and was lower than in watered controls. NSC decline resulted primarily from decreased sugar concentrations. Starch initially declined, and then increased above pre-drought concentrations before mortality. Although temperature did not affect NSC and sugar, starch concentrations ceased declining and increased earlier with higher temperatures. Reduced foliar NSC during lethal drought indicates a carbon metabolism role in mortality mechanism. Although carbohydrates were not completely exhausted at mortality, temperature differences in starch accumulation timing suggest that carbon metabolism changes are associated with time to death. Drought mortality appears to be related to temperature-dependent carbon dynamics concurrent with increasing hydraulic stress in P. edulis and potentially other similar species. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  16. Determination of water-extractable nonstructural carbohydrates, including inulin, in grass samples with high-performance anion exchange chromatography and pulsed amperometric detection.

    Science.gov (United States)

    Raessler, Michael; Wissuwa, Bianka; Breul, Alexander; Unger, Wolfgang; Grimm, Torsten

    2008-09-10

    The exact and reliable determination of carbohydrates in plant samples of different origin is of great importance with respect to plant physiology. Additionally, the identification and quantification of carbohydrates are necessary for the evaluation of the impact of these compounds on the biogeochemistry of carbon. To attain this goal, it is necessary to analyze a great number of samples with both high sensitivity and selectivity within a limited time frame. This paper presents a rugged and easy method that allows the isocratic chromatographic determination of 12 carbohydrates and sugar alcohols from one sample within 30 min. The method was successfully applied to a variety of plant materials with particular emphasis on perennial ryegrass samples of the species Lolium perenne. The method was easily extended to the analysis of the polysaccharide inulin after its acidic hydrolysis into the corresponding monomers without the need for substantial change of chromatographic conditions or even the use of enzymes. It therefore offers a fundamental advantage for the analysis of the complex mixture of nonstructural carbohydrates often found in plant samples.

  17. The role of matricellular proteins in glaucoma.

    LENUS (Irish Health Repository)

    Wallace, Deborah M

    2014-07-01

    Glaucoma is an optic neuropathy affecting approximately 60million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm\\'s canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.

  18. The influence of short-term cold stress on the metabolism of non-structural carbohydrates in polar grasses

    Directory of Open Access Journals (Sweden)

    Łopieńska-Biernat Elżbieta

    2017-06-01

    Full Text Available Plants adapt to extremely low temperatures in polar regions by maximizing their photosynthetic efficiency and accumulating cryoprotective and osmoprotective compounds. Flowering plants of the family Poaceae growing in the Arctic and in the Antarctic were investigated. Their responses to cold stress were analyzed under laboratory conditions. Samples were collected after 24 h and 48 h of cold treatment. Quantitative and qualitative changes of sugars are found among different species, but they can differ within a genus of the family Poaceae. The values of the investigated parameters in Poa annua differed considerably depending to the biogeographic origin of plants. At the beginning of the experiment, Antarctic plants were acclimatized in greenhouse characterized by significantly higher content of sugars, including storage reserves, sucrose and starch, but lower total protein content. After 24 h of exposure to cold stress, much smaller changes in the examined parameters were noted in Antarctic plants than in locally grown specimens. Total sugar content and sucrose, starch and glucose levels were nearly constant in P. annua, but they varied significantly. Those changes are responsible for the high adaptability of P. annua to survive and develop in highly unsupportive environments and colonize new regions.

  19. Morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways.

    Directory of Open Access Journals (Sweden)

    Senthil K Chinnakannan

    Full Text Available Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV and measles virus (MeV have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFNα/β and type II (IFNγ interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV, measles virus (MeV, peste des petits ruminants virus (PPRV and canine distemper virus (CDV. These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action.

  20. The sweet side of global change-dynamic responses of non-structural carbohydrates to drought, elevated CO2 and nitrogen fertilization in tree species.

    Science.gov (United States)

    Li, Weibin; Hartmann, Henrik; Adams, Henry D; Zhang, Hongxia; Jin, Changjie; Zhao, Chuanyan; Guan, Dexin; Wang, Anzhi; Yuan, Fenghui; Wu, Jiabing

    2018-06-11

    Non-structural carbohydrates (NSC) play a central role in plant functioning as energy carriers and building blocks for primary and secondary metabolism. Many studies have investigated how environmental and anthropogenic changes, like increasingly frequent and severe drought episodes, elevated CO2 and atmospheric nitrogen (N) deposition, influence NSC concentrations in individual trees. However, this wealth of data has not been analyzed yet to identify general trends using a common statistical framework. A thorough understanding of tree responses to global change is required for making realistic predictions of vegetation dynamics. Here we compiled data from 57 experimental studies on 71 tree species and conducted a meta-analysis to evaluate general responses of stored soluble sugars, starch and total NSC (soluble sugars + starch) concentrations in different tree organs (foliage, above-ground wood and roots) to drought, elevated CO2 and N deposition. We found that drought significantly decreased total NSC in roots (-17.3%), but not in foliage and above-ground woody tissues (bole, branch, stem and/or twig). Elevated CO2 significantly increased total NSC in foliage (+26.2%) and roots (+12.8%), but not in above-ground wood. By contrast, total NSC significantly decreased in roots (-17.9%), increased in above-ground wood (+6.1%), but was unaffected in foliage from N fertilization. In addition, the response of NSC to three global change drivers was strongly affected by tree taxonomic type, leaf habit, tree age and treatment intensity. Our results pave the way for a better understanding of general tree function responses to drought, elevated CO2 and N fertilization. The existing data also reveal that more long-term studies on mature trees that allow testing interactions between these factors are urgently needed to provide a basis for forecasting tree responses to environmental change at the global scale.

  1. Seasonal and inter-annual dynamics of growth, non-structural carbohydrates and C stable isotopes in a Mediterranean beech forest.

    Science.gov (United States)

    Scartazza, Andrea; Moscatello, Stefano; Matteucci, Giorgio; Battistelli, Alberto; Brugnoli, Enrico

    2013-07-01

    Seasonal and inter-annual dynamics of growth, non-structural carbohydrates (NSC) and carbon isotope composition (δ(13)C) of NSC were studied in a beech forest of Central Italy over a 2-year period characterized by different environmental conditions. The net C assimilated by forest trees was mainly used to sustain growth early in the season and to accumulate storage carbohydrates in trunk and root wood in the later part of the season, before leaf shedding. Growth and NSC concentration dynamics were only slightly affected by the reduced soil water content (SWC) during the drier year. Conversely, the carbon isotope analysis on NSC revealed seasonal and inter-annual variations of photosynthetic and post-carboxylation fractionation processes, with a significant increase in δ(13)C of wood and leaf soluble sugars in the drier summer year than in the wetter one. The highly significant correlation between δ(13)C of leaf soluble sugars and SWC suggests a decrease of the canopy C isotope discrimination and, hence, an increased water-use efficiency with decreasing soil water availability. This may be a relevant trait for maintaining an acceptable plant water status and a relatively high C sink capacity during dry seasonal periods. Our results suggest a short- to medium-term homeostatic response of the Collelongo beech stand to variations in water availability and solar radiation, indicating that this Mediterranean forest was able to adjust carbon-water balance in order to prevent C depletion and to sustain plant growth and reserve accumulation during relatively dry seasons.

  2. Potential of semi-structural and non-structural adaptation strategies to reduce future flood risk: case study for the Meuse

    Directory of Open Access Journals (Sweden)

    J. K. Poussin

    2012-11-01

    Full Text Available Flood risk throughout Europe has increased in the last few decades, and is projected to increase further owing to continued development in flood-prone areas and climate change. In recent years, studies have shown that adequate undertaking of semi-structural and non-structural measures can considerably decrease the costs of floods for households. However, there is little insight into how such measures can decrease the risk beyond the local level, now and in the future. To gain such insights, a modelling framework using the Damagescanner model with land-use and inundation maps for 2000 and 2030 was developed and applied to the Meuse river basin, in the region of Limburg, in the southeast of the Netherlands. The research suggests that annual flood risk may increase by up to 185% by 2030 compared with 2000, as a result of combined land-use and climate changes. The independent contributions of climate change and land-use change to the simulated increase are 108% and 37%, respectively. The risk-reduction capacity of the implementation of spatial zoning measures, which are meant to limit and regulate developments in flood-prone areas, is between 25% and 45%. Mitigation factors applied to assess the potential impact of three mitigation strategies (dry-proofing, wet-proofing, and the combination of dry- and wet-proofing in residential areas show that these strategies have a risk-reduction capacity of between 21% and 40%, depending on their rate of implementation. Combining spatial zoning and mitigation measures could reduce the total increase in risk by up to 60%. Policy implications of these results are discussed. They focus on the undertaking of effective mitigation measures, and possible ways to increase their implementation by households.

  3. Modulation of carbon and nitrogen allocation in Urtica dioica and Plantago major by elevated CO{sub 2}. Impact of accumulation of nonstructural carbohydrates and ontogenetic drift

    Energy Technology Data Exchange (ETDEWEB)

    Hertog, J. den; Stulen, I.; Fonseca, F.; Delea, P.

    1996-10-01

    Doubling the atmospheric CO{sub 2} concentration from 350 to 700 {mu} l{sup -1} increased the relative growth rate (RGR) of hydroponically grown Urtica dioica L. and Plantagomajor ssp. pleiospherma Pilger only for the first 10-14 days. Previous experiments with P. major indicated that RGR did not respond i proportion to the rate of photosynthesis. The impact of changes in leaf morphology, dry matter partitioning, dry matter chemical composition and ontogenetic drift on this discrepancy is analysed. Soon after the start of the treatment, carbohydrate concentrations were higher at elevated CO{sub 2}; largely due to starch accumulation. An increase in the percentage of leaf dry matter and decreases in the specific leaf area (SLA) and the shoot nitrogen concentration were correlated with an increase in the total nonstructural carbohydrate concentration (TNC). A combination of accumulation of soluble sugars and starch and ontogenetic drift explains the decrease in SLA at the elevated CO{sub 2} level. A similar ontogenetic effect of elevated CO{sub 2} was observed on the specific root length (SRL). Shoot nitrogen concentration and percentage leaf dry matter were not affected. The net diurnal fluctuation of the carbohydrate pool in P. major was equal for both CO{sub 2} concentrations, indicating that the growth response to elevated CO{sub 2} may be ruled by other variables such as sink strength. Elevated CO{sub 2} did not greatly influence the partitioning of nitrogen between soluble and insoluble, reduced N and nitrate, nor the allocation of dry matter between leaf, stem and root. That the root to shoot ratio (F/S) was not affected by elevated CO{sub 2} implies that, to maintain a balanced activity between roots and shoot, no shift in partitioning of dry matter upon doubling of the atmospheric CO{sub 2} concentration is required. (AB)

  4. The effectiveness of the Pilates method: reducing the degree of non-structural scoliosis, and improving flexibility and pain in female college students.

    Science.gov (United States)

    Alves de Araújo, Maria Erivânia; Bezerra da Silva, Elirez; Bragade Mello, Danielli; Cader, Samária Ali; Shiguemi Inoue Salgado, Afonso; Dantas, Estélio Henrique Martin

    2012-04-01

    To evaluate the effectiveness of Pilates with regard to the degree of scoliosis, flexibility and pain. The study included 31 female students divided into two groups: a control group (CG = 11), which had no therapeutic intervention, and an experimental group (EG = 20), which underwent Pilates-based therapy. We used radiological goniometry measurements to assess the degree of scoliosis, standard goniometry measurements to determine the degree of flexibility and the scale of perceived pain using the Borg CR 10 to quantify the level of pain. The independent t test of the Cobb angle (t = - 2.317, p = 0.028), range of motion of trunk flexion (t = 3.088, p = 0.004) and pain (t = -2.478, p = 0.019) showed significant differences between the groups, with best values in the Pilates group. The dependent t test detected a significant decrease in the Cobb angle (Δ% = 38%, t = 6.115, p = 0.0001), a significant increase in trunk flexion (Δ% = 80%, t = -7.977, p = 0.0001) and a significant reduction in pain (Δ% = 60%, t = 7.102, p = 0.0001) in the EG. No significant difference in Cobb angle (t = 0.430, p = 0.676), trunk flexion, (t = 0.938p = 0.371) or pain (t = 0.896, p = 0.391) was found for the CG. The Pilates group was better than control group. The Pilates method showed a reduction in the degree of non-structural scoliosis, increased flexibility and decreased pain. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction.

    Science.gov (United States)

    Renukaradhya, Gourapura J; Meng, Xiang-Jin; Calvert, Jay G; Roof, Michael; Lager, Kelly M

    2015-08-07

    Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork

  6. Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E

    DEFF Research Database (Denmark)

    Chagnon, F; Lamarre, A; Lachance, C

    1998-01-01

    to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice......Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression...... and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used...

  7. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. The discrete ordinate method in association with the finite-volume method in non-structured mesh; Methode des ordonnees discretes associee a la methode des volumes finis en maillage non structure

    Energy Technology Data Exchange (ETDEWEB)

    Le Dez, V; Lallemand, M [Ecole Nationale Superieure de Mecanique et d` Aerotechnique (ENSMA), 86 - Poitiers (France); Sakami, M; Charette, A [Quebec Univ., Chicoutimi, PQ (Canada). Dept. des Sciences Appliquees

    1997-12-31

    The description of an efficient method of radiant heat transfer field determination in a grey semi-transparent environment included in a 2-D polygonal cavity with surface boundaries that reflect the radiation in a purely diffusive manner is proposed, at the equilibrium and in radiation-conduction coupling situation. The technique uses simultaneously the finite-volume method in non-structured triangular mesh, the discrete ordinate method and the ray shooting method. The main mathematical developments and comparative results with the discrete ordinate method in orthogonal curvilinear coordinates are included. (J.S.) 10 refs.

  9. The discrete ordinate method in association with the finite-volume method in non-structured mesh; Methode des ordonnees discretes associee a la methode des volumes finis en maillage non structure

    Energy Technology Data Exchange (ETDEWEB)

    Le Dez, V.; Lallemand, M. [Ecole Nationale Superieure de Mecanique et d`Aerotechnique (ENSMA), 86 - Poitiers (France); Sakami, M.; Charette, A. [Quebec Univ., Chicoutimi, PQ (Canada). Dept. des Sciences Appliquees

    1996-12-31

    The description of an efficient method of radiant heat transfer field determination in a grey semi-transparent environment included in a 2-D polygonal cavity with surface boundaries that reflect the radiation in a purely diffusive manner is proposed, at the equilibrium and in radiation-conduction coupling situation. The technique uses simultaneously the finite-volume method in non-structured triangular mesh, the discrete ordinate method and the ray shooting method. The main mathematical developments and comparative results with the discrete ordinate method in orthogonal curvilinear coordinates are included. (J.S.) 10 refs.

  10. Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication.

    Science.gov (United States)

    Fang, Jianyu; Wang, Haiyan; Bai, Juan; Zhang, Qiaoya; Li, Yufeng; Liu, Fei; Jiang, Ping

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13-16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection.

  11. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-09-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

  12. Nonstructural carbon dynamics are best predicted by the combination of photosynthesis and plant hydraulics during both bark beetle induced mortality and herbaceous plant response to drought

    Science.gov (United States)

    Ewers, B. E.; Mackay, D. S.; Guadagno, C.; Peckham, S. D.; Pendall, E.; Borkhuu, B.; Aston, T.; Frank, J. M.; Massman, W. J.; Reed, D. E.; Yarkhunova, Y.; Weinig, C.

    2012-12-01

    Recent work has shown that nonstructural carbon (NSC) provides both a signal and consequence of water stress in plants. The dynamics of NSC are likely not solely a result of the balance of photosynthesis and respiration (carbon starvation hypothesis) but also the availability of NSC for plant functions due to hydraulic condition. Further, plant hydraulics regulates photosynthesis both directly through stomatal conductance and indirectly through leaf water status control over leaf biochemistry. To test these hypotheses concerning NSC in response to a wide variety of plant perturbations, we used a model that combines leaf biochemical controls over photosynthesis (Farquhar model) with dynamic plant hydraulic conductance (Sperry model). This model (Terrestrial Regional Ecosystem Exchange Simulator; TREES) simulates the dynamics of NSC through a carbon budget approach that responds to plant hydraulic status. We tested TREES on two dramatically different datasets. The first dataset is from lodgepole pine and Engelmann spruce trees dying from bark beetles that carry blue-stain fungi which block xylem and cause hydraulic failure. The second data set is from Brassica rapa, a small herbaceous plant whose accessions are used in a variety of crops. The Brassica rapa plants include two parents whose circadian clock periods are different; NSC is known to provide inputs to the circadian clock likely modified by drought. Thus, drought may interact with clock control to constrain how NSC changes over the day. The Brassica rapa plants were grown in growth chamber conditions where drought was precisely controlled. The connection between these datasets is that both provide rigorous tests of our understanding of plant NSC dynamics and use similar leaf and whole plant gas exchange and NSC laboratory methods. Our results show that NSC decline (water stress. The model is able to capture this relatively small decline in NSC by limiting NSC utilization through loss of plant hydraulic

  13. Interannual and seasonal dynamics, and the age, of nonstructural carbohydrate pools in the stemwood of temperate trees across a climatic gradient in New England

    Science.gov (United States)

    Richardson, A. D.; Carbone, M. S.; Czimczik, C. I.; Keenan, T. F.; Schaberg, P.; Xu, X.

    2011-12-01

    Like all plants, forest trees accumulate and store surplus mobile carbon (C) compounds as resources to be used to support future growth. This can be viewed as a bet-hedging strategy, providing reserves that the tree can draw on in times of stress-e.g., following disturbance, disease, or extreme climatic events. In the context of climate change, understanding factors influencing the availability of these stored C compounds to support growth and metabolism is essential for predicting the resilience of forests to environmental stress factors. We conducted this study to investigate the role of these stored C pools in the context ecosystem C balance at time scales from days to years. At quarterly intervals over a three year period, we monitored stemwood total nonstructural carbohydrate (TNC) concentrations of the dominant tree species of New England. Work was conducted at three sites along a climatic gradient: an oak-dominated transition hardwood forest (Harvard Forest), a maple-beech-birch northern hardwood forest (Bartlett Experimental Forest), and a spruce-fir forest (Howland Forest). We observed large differences among species both in TNC concentrations, and in how the TNC pool is partitioned to different compounds (starch, sucrose, glucose, fructose, raffinose, xylose and stachyose). Within a species, however, seasonal dynamics were remarkably similar across sites. The interannual variability in maximum TNC concentrations appears to be smaller than interannual variability in annual net ecosystem exchange of CO2. With an additional set of samples, we are using the bomb radiocarbon (14C) spike to estimate the average age of the sugars and starches in the TNC pool, and relating this to factors such as size, age, and recent growth rates of each tree. Initial results suggest that these TNC pools range in age from several years to several decades old. The average ages of starch and sugar pools are related, with the starches generally being older than sugars

  14. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  15. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.

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    Dorothee A Vogt

    Full Text Available The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web and assists viral assembly in the close vicinity of lipid droplets (LDs. To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1-31, a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47 as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon, indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47--via its interaction with NS5A--serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.

  16. A systematic review of structured versus non-structured breastfeeding programmes to support the initiation and duration of exclusive breastfeeding in acute and primary healthcare settings.

    Science.gov (United States)

    Beake, Sarah; Pellowe, Carol; Dykes, Fiona; Schmied, Virginia; Bick, Debra

    2011-01-01

    Background: Breastfeeding has many important health benefits for the woman and her baby. Despite evidence of benefit from a large number of well conducted studies, breastfeeding uptake and the duration of exclusive breastfeeding remain low in many countries. In order to improve breastfeeding rates, policy and guidelines at global, individual country level and in local healthcare settings have recommended that structured programmes to support breastfeeding should be introduced. The objective of this review was to consider the evidence of outcomes of structured compared with non-structured breastfeeding programmes in acute maternity care settings to support initiation and duration of exclusive breastfeeding. The definition of structured programme used included a multi-faceted or single intervention approach to support breastfeeding; definition of non-structured included support offered within standard care. The review considered quantitative and qualitative studies which addressed outcomes following the introduction of a structured programme in acute healthcare settings to support breastfeeding compared with no programme. The primary outcomes of interest were uptake of breastfeeding and duration of exclusive breastfeeding (only breast milk, including milk expressed). Studies which only considered community based interventions were not included. A search of the literature published between 1992 and 2010 was conducted, which followed a four step process. After a limited search of MEDLINE and CINAHL to identify key words contained in the title or abstract and index terms to describe relevant interventions, a second extensive search was undertaken using identified key words and index terms. The third step included a search of reference lists and bibliographies of relevant articles and the fourth step included a search of grey and unpublished literature and national databasesMethodological quality: Methodological quality was assessed using checklists developed by the

  17. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

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    Albrecht von Brunn

    2007-05-01

    Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

  18. Evaluation of certain crop residues for carbohydrate and protein fractions by cornell net carbohydrate and protein system

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    Venkateswarulu Swarna

    2015-06-01

    Full Text Available Four locally available crop residues viz., jowar stover (JS, maize stover (MS, red gram straw (RGS and black gram straw (BGS were evaluated for carbohydrate and protein fractions using Cornell Net Carbohydrate and Protein (CNCP system. Lignin (% NDF was higher in legume straws as compared to cereal stovers while Non-structural carbohydrates (NSC (% DM followed the reverse trend. The carbohydrate fractions A and B1 were higher in BGS while B2 was higher in MS as compared to other crop residues. The unavailable cell wall fraction (C was higher in legume straws when compared to cereal stovers. Among protein fractions, B1 was higher in legume straws when compared to cereal stovers while B2 was higher in cereal stovers as compared to legume straws. Fraction B3 largely, bypass protein was highest in MS as compared to other crop residues. Acid detergent insoluble crude protein (ADICP (% CP or unavailable protein fraction C was lowest in MS and highest in BGS. It is concluded that MS is superior in nutritional value for feeding ruminants as compared to other crop residues.

  19. Systematic analysis of protein identity between Zika virus and other arthropod-borne viruses.

    Science.gov (United States)

    Chang, Hsiao-Han; Huber, Roland G; Bond, Peter J; Grad, Yonatan H; Camerini, David; Maurer-Stroh, Sebastian; Lipsitch, Marc

    2017-07-01

    To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains. We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k -mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 k -mers) of protein fragments on the list. We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections.

  20. Identification of a Novel Recombinant Type 2 Porcine Reproductive and Respiratory Syndrome Virus in China

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    Long Zhou

    2018-03-01

    Full Text Available Since the emergence of NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV in China in 2013, PRRSVs have undergone rapid evolution. In this study, a novel variant of PRRSV strain (designated SCcd17 was successfully isolated from piglets with clinical signs in Sichuan Province in China in 2017, and the complete genomic sequence was determined. The genome of this new isolate was 15,015 nucleotides (nt long, and comparative analysis revealed that SCcd17 exhibited 90.2%, 85.2%, 84.9%, and 84.0% nucleotide similarity to PRRSVs NADC30, JXA1, CH-1a, and VR-2332, respectively. Phylogenetic analysis indicated that the SCcd17 strain was classified into the NADC30-like sub-genotype, in which all the strains contained the unique discontinuous 131-amino acid deletion in nonstructural protein 2 (nsp2 when compared to VR-2332-like viruses. Notably, extensive amino acid substitutions were observed in nsp2 and a unique single amino acid deletion at position 33 of the GP5 is being described for the first time. Strikingly, recombination analysis revealed that SCcd17 was the result of recombination between the NADC30-like, JXA1-like, and VR-2332-like strains at five recombination breakpoints: nsp1α (nt 641, nsp3 (nt 5141, nsp10 (nt 9521, open reading frame 3 (ORF3 (nt 12,581, and ORF4 (nt 13,021. The genomic data of SCcd17 will be helpful for understanding the role of genomic recombination in the evolution of PRRSV.

  1. A global analysis of the concentration and dynamics of non-structural carbohydrates in plants: does it matter under global change? (Invited)

    Science.gov (United States)

    Sala, A.; Martínez-Vilalta, J.; Asencio, M.; Lloret, F.; Palacio, S.; Galiano, L.; Hoch, G.; Piper, F.

    2013-12-01

    Forests store significant amounts of C globally and recent reports of forest mortality world-wide have generated strong concern. Evidence suggests that increasing drought associated with climate change is a primary cause of tree stress and subsequent mortality. This has generated an urgent need to predict how forests will cope with increasing stress. Storage of non-structural C compounds (NSC, compounds not permanently invested in structural biomass that can later be used to support diverse plant functions) is critical for survival during periods when C assimilation does not meet demand. However, remarkable knowledge gaps exist to accurately predict plant growth and survival under climate change. Although trees accumulate relatively large pools of NSC, there is a strong debate on how these pools build up over time. On the one hand, it is frequently assumed that the build- up of NSC in trees occurs when supply via photosynthesis exceeds overall demands. If so, the abundant NSC pools in trees reflect an overabundance of C in the long term. An alternative explanation is that trees regulate NSC storage to maintain sufficient pools to cope with asynchronies between demand and supply and with stresses that long lived plants inevitably experience during their life time. However, our understanding of whether and how trees regulate storage in the long term is minimal. Here, we assembled a new global database to examine broad patterns of seasonal NSC variation across organs, life forms and biomes, and the degree to which NSC storage is depleted in plants under a wide range of natural conditions. We compiled seasonal data (at least three measurements over a minimum of four months) for ca. 200 wild species under natural conditions. On average, NSC account for ca. 8-10% of dry plant biomass. NSC and starch concentrations do not vary significantly with biome, but soluble sugars (SS) in plants from Mediterranean biomes are higher than in temperate or tropical biomes. On average

  2. A Structural Perspective on the Modulation of Protein-Protein Interactions with Small Molecules.

    Science.gov (United States)

    Demirel, Habibe Cansu; Dogan, Tunca; Tuncbag, Nurcan

    2018-05-31

    Protein-protein interactions (PPIs) are the key components in many cellular processes including signaling pathways, enzymatic reactions and epigenetic regulation. Abnormal interactions of some proteins may be pathogenic and cause various disorders including cancer and neurodegenerative diseases. Although inhibiting PPIs with small molecules is a challenging task, it gained an increasing interest because of its strong potential for drug discovery and design. The knowledge of the interface as well as the structural and chemical characteristics of the PPIs and their roles in the cellular pathways are necessary for a rational design of small molecules to modulate PPIs. In this study, we review the recent progress in the field and detail the physicochemical properties of PPIs including binding hot spots with a focus on structural methods. Then, we review recent approaches for structural prediction of PPIs. Finally, we revisit the concept of targeting PPIs in a systems biology perspective and we refer to the non-structural approaches, usually employed when the structural information is not present. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus

    International Nuclear Information System (INIS)

    Rowland, Raymond R.R.; Schneider, Paula; Fang Ying; Wootton, Sarah; Yoo, Dongwan; Benfield, David A.

    2003-01-01

    The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-α, importin-β nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus

  4. Protein composition of the hepatitis A virus quasi-envelope.

    Science.gov (United States)

    McKnight, Kevin L; Xie, Ling; González-López, Olga; Rivera-Serrano, Efraín E; Chen, Xian; Lemon, Stanley M

    2017-06-20

    The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins ( n = 105) were significantly enriched for components of the endolysosomal system (>60%, P hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.

  5. Evaluation of energy status of dairy cows using milk fat, protein and urea concentrations

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    Kirovski Danijela

    2011-11-01

    Full Text Available Energy status of dairy cows may be estimated using results for concentrations of fat, protein and urea (MUN in milk samples obtained from bulk tank or individual cows. Using individual cow milk samples is recommended on dairy farms in our geografical region due to the unhomogenity of cows in the herds in respect to their genetic potential for milk production. Depression of milk fat occurs as a consequence of heat stress, underfeeding of peripartal cows, overfeeding concentrate with reduced ration fiber levels or overfeeding with dietary fat. High milk fat content is usually combined with severe negative energy balance. Nutrition and feeding practices have great impact on milk protein level. A deficiency of crude protein in the ration may depress protein in milk. Feeding excessive dietary protein does not significantly increase milk protein. MUN analyses point out potential problems in feeding program on dairy farm. High MUN values may reflect excessive dietary crude protein and/or low rumen degradable non fiber carbohydrates intake. Also, MUN levels is impacted by heat stress since its value is increased during the summer season. Low MUNs indicate a possible dietary protein deficiency. Additionally, low MUNs concentration may indicate excess in dietary nonstructural carbohydrates. On the bases on the interrelationships between protein and urea concentrations, as well as protein and fat concentrations in individual milk sample, estimation of energy balance of dairy cows may be done more accurately.

  6. A PCV2 vaccine based on genotype 2b is more effective than a 2a-based vaccine to protect against PCV2b or combined PCV2a/2b viremia in pigs with concurrent PCV2, PRRSV and PPV infection.

    Science.gov (United States)

    Opriessnig, Tanja; O'Neill, Kevin; Gerber, Priscilla F; de Castro, Alessandra M M G; Gimenéz-Lirola, Luis G; Beach, Nathan M; Zhou, Lei; Meng, Xiang-Jin; Wang, Chong; Halbur, Patrick G

    2013-01-07

    The predominant genotype of porcine circovirus (PCV) in the pig population today is PCV2b yet PCV2a-based commercial vaccines are considered effective in protecting against porcine circovirus associated disease. The objective of this study was to compare the ability of PCV2a- and PCV2b-based vaccines to control PCV2b viremia in a challenge model that mimics the U.S. field situation. Sixty-three pigs were randomly assigned to one of eight groups. Sixteen pigs were vaccinated with an experimental live-attenuated chimeric PCV1-2a vaccine based on genotype 2a and another 16 pigs with a chimeric PCV1-2b vaccine based on genotype 2b. Challenge was done 28 days post vaccination (dpv) using PCV2b (or a combination of PCV2a and PCV2b), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV) to mimic what commonly occurs in the field. The experiment was terminated 21 days post challenge (dpc) or 49dpv. Pigs vaccinated with the chimeric PCV1-2b vaccine had significantly higher levels of PCV1-2b viremia and shedding of the PCV1-2b vaccine virus in feces and nasal secretions but also a more robust humoral immune response as evidenced by significantly higher ELISA S/P ratios compared to the PCV1-2a vaccination. Regardless of challenge, the PCV1-2b vaccination significantly reduced the prevalence and amount of PCV2 viremia compared to the PCV1-2a vaccination. Interestingly, in the non-vaccinated pigs concurrent PCV2a infection resulted in clinical disease and increased macroscopic lung lesions compared to pigs challenged with PCV2b alone, further supporting the idea that concurrent PCV2a/PCV2b infection is necessary for optimal PCV2 replication. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tokyo.ac.jp; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-15

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.

  8. Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

    Science.gov (United States)

    Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

    2015-01-01

    Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

    OpenAIRE

    Vanacker, J M; Corbau, R; Adelmant, G; Perros, M; Laudet, V; Rommelaere, J

    1996-01-01

    The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the...

  10. The Influenza NS1 Protein: What Do We Know in Equine Influenza Virus Pathogenesis?

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2016-08-01

    Full Text Available Equine influenza virus remains a serious health and potential economic problem throughout most parts of the world, despite intensive vaccination programs in some horse populations. The influenza non-structural protein 1 (NS1 has multiple functions involved in the regulation of several cellular and viral processes during influenza infection. We review the strategies that NS1 uses to facilitate virus replication and inhibit antiviral responses in the host, including sequestering of double-stranded RNA, direct modulation of protein kinase R activity and inhibition of transcription and translation of host antiviral response genes such as type I interferon. Details are provided regarding what it is known about NS1 in equine influenza, especially concerning C-terminal truncation. Further research is needed to determine the role of NS1 in equine influenza infection, which will help to understand the pathophysiology of complicated cases related to cytokine imbalance and secondary bacterial infection, and to investigate new therapeutic and vaccination strategies.

  11. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

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    Marcio Hedil

    2016-07-01

    Full Text Available The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  12. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    Science.gov (United States)

    Hedil, Marcio; Kormelink, Richard

    2016-07-23

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  13. Analysis of the PDZ binding specificities of Influenza A Virus NS1 proteins

    Directory of Open Access Journals (Sweden)

    Nagasaka Kazunori

    2011-01-01

    Full Text Available Abstract The Influenza A virus non-structural protein 1 (NS1 is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant. In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.

  14. The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/ MDA5 activation.

    Science.gov (United States)

    Ding, Zhen; Fang, Liurong; Yuan, Shuangling; Zhao, Ling; Wang, Xunlei; Long, Siwen; Wang, Mohan; Wang, Dang; Foda, Mohamed Frahat; Xiao, Shaobo

    2017-07-25

    Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-β production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N-PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-β production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.

  15. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  16. Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-β antagonism and protein stability.

    Science.gov (United States)

    Gori Savellini, Gianni; Gandolfo, Claudia; Cusi, Maria Grazia

    2015-12-01

    Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Detecting Molecular Features of Spectra Mainly Associated with Structural and Non-Structural Carbohydrates in Co-Products from BioEthanol Production Using DRIFT with Uni- and Multivariate Molecular Spectral Analyses

    Science.gov (United States)

    Yu, Peiqiang; Damiran, Daalkhaijav; Azarfar, Arash; Niu, Zhiyuan

    2011-01-01

    The objective of this study was to use DRIFT spectroscopy with uni- and multivariate molecular spectral analyses as a novel approach to detect molecular features of spectra mainly associated with carbohydrate in the co-products (wheat DDGS, corn DDGS, blend DDGS) from bioethanol processing in comparison with original feedstock (wheat (Triticum), corn (Zea mays)). The carbohydrates related molecular spectral bands included: A_Cell (structural carbohydrates, peaks area region and baseline: ca. 1485–1188 cm−1), A_1240 (structural carbohydrates, peak area centered at ca. 1240 cm−1 with region and baseline: ca. 1292–1198 cm−1), A_CHO (total carbohydrates, peaks region and baseline: ca. 1187–950 cm−1), A_928 (non-structural carbohydrates, peak area centered at ca. 928 cm−1 with region and baseline: ca. 952–910 cm−1), A_860 (non-structural carbohydrates, peak area centered at ca. 860 cm−1 with region and baseline: ca. 880–827 cm−1), H_1415 (structural carbohydrate, peak height centered at ca. 1415 cm−1 with baseline: ca. 1485–1188 cm−1), H_1370 (structural carbohydrate, peak height at ca. 1370 cm−1 with a baseline: ca. 1485–1188 cm−1). The study shows that the grains had lower spectral intensity (KM Unit) of the cellulosic compounds of A_1240 (8.5 vs. 36.6, P carbohydrate of A_928 (17.3 vs. 2.0) and A_860 (20.7 vs. 7.6) than their co-products from bioethanol processing. There were no differences (P > 0.05) in the peak area intensities of A_Cell (structural CHO) at 1292–1198 cm−1 and A_CHO (total CHO) at 1187–950 cm−1 with average molecular infrared intensity KM unit of 226.8 and 508.1, respectively. There were no differences (P > 0.05) in the peak height intensities of H_1415 and H_1370 (structural CHOs) with average intensities 1.35 and 1.15, respectively. The multivariate molecular spectral analyses were able to discriminate and classify between the corn and corn DDGS molecular spectra, but not wheat and wheat DDGS. This

  18. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  19. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  20. Whey Protein

    Science.gov (United States)

    ... reliable information about the safety of taking whey protein if you are pregnant or breast feeding. Stay on the safe side and avoid use. Milk allergy: If you are allergic to cow's milk, avoid using whey protein.

  1. Identification of adaptive mutations in the influenza A virus non-structural 1 gene that increase cytoplasmic localization and differentially regulate host gene expression.

    Directory of Open Access Journals (Sweden)

    Nicole Forbes

    Full Text Available The NS1 protein of influenza A virus (IAV is a multifunctional virulence factor. We have previously characterized gain-of-function mutations in the NS1 protein arising from the experimental adaptation of the human isolate A/Hong Kong/1/1968(H3N2 (HK to the mouse. The majority of these mouse adapted NS1 mutations were demonstrated to increase virulence, viral fitness, and interferon antagonism, but differ in binding to the post-transcriptional processing factor cleavage and polyadenylation specificity factor 30 (CPSF30. Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. In human cells the HK wild-type (HK-wt virus NS1 protein partitioned equivalently between the cytoplasm and nucleus but was defective in cytoplasmic localization in mouse cells. Several adaptive mutations increased the proportion of NS1 in the cytoplasm of mouse cells with the greatest effects for mutations M106I and D125G. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low extents of host-gene regulation (HGR or LGR phenotypes. While host genes were predominantly down regulated for the HGR group of mutants (D2N, V23A, F103L, M106I+L98S, L98S, M106V, and M106V+M124I, the LGR phenotype mutants (D125G, M106I, V180A, V226I, and R227K were characterized by a predominant up regulation of host genes. CPSF30 binding affinity of NS1 mutants did not predict effects on host gene expression. To our knowledge this is the first report of roles of adaptive NS1 mutations that impact intracellular localization and regulation of host gene expression.

  2. Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: The possible link between carbon starvation and hydraulic failure.

    Science.gov (United States)

    Trifilò, Patrizia; Casolo, Valentino; Raimondo, Fabio; Petrussa, Elisa; Boscutti, Francesco; Lo Gullo, Maria Assunta; Nardini, Andrea

    2017-11-01

    Drought-induced tree decline is a complex event, and recent hypotheses suggest that hydraulic failure and carbon starvation are co-responsible for this process. We tested the possible role of non-structural carbohydrates (NSC) content on post-drought hydraulic recovery, to verify the hypothesis that embolism reversal represents a mechanistic link between carbon starvation and stem hydraulics. Measurements were performed in laurel plants subjected to similar water stress levels either over short or long term, to induce comparable embolism levels. Plants subjected to mild and prolonged water shortage (S) showed reduced growth, adjustment of turgor loss point driven by changes in both osmotic potential at full turgor and bulk modulus of elasticity, a lower content of soluble NSC and a higher content of starch with respect to control (C) plants. Moreover, S plants showed a lower ability to recover from xylem embolism than C plants, even after irrigation. Our data suggest that plant carbon status might indirectly influence plant performance during and after drought via effects on xylem hydraulic functioning, supporting the view of a possible mechanistic link between the two processes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Role of Bovine Adenovirus-3 33K protein in viral replication

    International Nuclear Information System (INIS)

    Kulshreshtha, Vikas; Babiuk, Lorne A.; Tikoo, Suresh K.

    2004-01-01

    The L6 region of bovine adenovirus type (BAdV)-3 encodes a nonstructural protein named 33K. To identify and characterize the 33K protein, rabbit polyclonal antiserum was raised against a 33K-GST fusion protein expressed in bacteria. Anti-33K serum immunoprecipitated a protein of 42 kDa in in vitro translated and transcribed mRNA of 33K. However, three proteins of 42, 38, and 33 kDa were detected in BAdV-3 infected cells. To determine the role of this protein in virus replication, a recombinant BAV-33S1 containing insertional inactivation of 33K (a stop codon created at the seventh amino acid of 33K ORF) was constructed. Although BAV-33S1 could be isolated, the mutant showed a severe defect in the production of progeny virus. Inactivation of the 33K gene showed no effect on early and late viral gene expression in cells infected with BAV-33S1. However, formation of mature virions was significantly reduced in cells infected with BAV-33S1. Surprisingly, insertional inactivation of 33K at amino acid 97 (pFBAV-33.KS2) proved lethal for virus production. Although expression of early or late genes was not affected, no capsid formation could be observed in mutant DNA-transfected cells. These results suggest that 33K is required for capsid assembly and efficient DNA capsid interaction

  4. Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein

    Science.gov (United States)

    Fassbinder-Orth, C. A.; Hofmeister, Erik K.; Weeks-Levy, C.; Karasov, W.H.

    2009-01-01

    West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 ??g or 100 ??g/dose), of E protein intramuscularly (IM, 20 ??g/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds. ?? 2009 American Association of Avian Pathologists.

  5. Role of the NSs protein in the zoonotic capacity of Orthobunyaviruses.

    Science.gov (United States)

    Hart, T J; Kohl, A; Elliott, R M

    2009-08-01

    The family Bunyaviridae contains over 350 named isolates, classified into five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The Orthobunyavirus genus contains some 170 isolates that are mainly transmitted by mosquitoes and are responsible for a range of disease syndromes in humans including self-limiting febrile illness, encephalitis and haemorrhagic fever. The viruses have a tripartite, negative-sense RNA genome. Analyses of viruses in four serogroups (Bunyamwera, California, Group C and Simbu) showed that the smallest (S) RNA segment encodes the nucleocapsid protein (N) and a non-structural protein called (NSs). The NSs protein of Bunyamwera virus (BUNV) has been shown to play a role in shut-off of host cell protein synthesis in mammalian cells, but no protein shut-off is observed in BUNVinfected mosquito cells (Aedes albopictus C6/36 cells). Protein shut-off in infected mammalian cells is achieved by global inhibition of RNA polymerase II-mediated transcription and enables the virus to overcome the host innate immune response. As innate defence mechanisms constitute a significant barrier to virus infection of different hosts, NSs would appear to play a key role in determining the zoonotic capacity of orthobunyaviruses.

  6. Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

    Directory of Open Access Journals (Sweden)

    Xiaoyao Yang

    2017-07-01

    Full Text Available Among the few non-structural proteins encoded by the picornaviral genome, the 2A protein is particularly special, irrespective of structure or function. During the evolution of the Picornaviridae family, the 2A protein has been highly non-conserved. We believe that the 2A protein in this family can be classified into at least five distinct types according to previous studies. These five types are (A chymotrypsin-like 2A, (B Parechovirus-like 2A, (C hepatitis-A-virus-like 2A, (D Aphthovirus-like 2A, and (E 2A sequence of the genus Cardiovirus. We carried out a phylogenetic analysis and found that there was almost no homology between each type. Subsequently, we aligned the sequences within each type and found that the functional motifs in each type are highly conserved. These different motifs perform different functions. Therefore, in this review, we introduce the structures and functions of these five types of 2As separately. Based on the structures and functions, we provide suggestions to combat picornaviruses. The complexity and diversity of the 2A protein has caused great difficulties in functional and antiviral research. In this review, researchers can find useful information on the 2A protein and thus conduct improved antiviral research.

  7. Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

    Science.gov (United States)

    Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

    2017-09-15

    Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

  8. 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.

  9. Bam35 tectivirus intraviral interaction map unveils new function and localization of phage ORFan proteins.

    Science.gov (United States)

    Berjón-Otero, Mónica; Lechuga, Ana; Mehla, Jitender; Uetz, Peter; Salas, Margarita; Redrejo-Rodríguez, Modesto

    2017-07-26

    Tectiviridae comprises a group of tail-less, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria.In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing the knowledge of the viral proteome, we carried out a comprehensive yeast two-hybrid analysis among all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprises 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggested that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, being the latter also supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving non-structural proteins, such as the DNA binding protein P1 and the genome terminal protein (P4), which was confirmed by co-immunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle. IMPORTANCE Tail-less viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown

  10. Identification of cross-reacting T-cell epitopes in structural and non-structural proteins of swine and pandemic H1N1 influenza A virus strains in pigs

    DEFF Research Database (Denmark)

    Baratelli, Massimiliano; Pedersen, Lasse Eggers; Trebbien, Ramona

    2017-01-01

    Heterologous protection against swine influenza viruses (SwIVs) of different lineages is an important concern for the pig industry. Cross-protection between 'avian-like' H1N1 and 2009 pandemic H1N1 lineages has been observed previously, indicating the involvement of cross-reacting T-cells. Here...

  11. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    International Nuclear Information System (INIS)

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-01-01

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  12. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication.

    Directory of Open Access Journals (Sweden)

    Mónica González-Magaldi

    Full Text Available Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2 that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7 sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.

  13. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

  14. Cloning, purification and preliminary crystallographic studies of the 2AB protein from hepatitis A virus

    International Nuclear Information System (INIS)

    Garriga, Damià; Vives-Adrián, Laia; Buxaderas, Mònica; Ferreira-da-Silva, Frederico; Almeida, Bruno; Macedo-Ribeiro, Sandra; Pereira, Pedro José Barbosa; Verdaguer, Núria

    2011-01-01

    The 2AB protein derived from the nonstructural P2 region of hepatitis A virus has been cloned, purified and crystallized. The preliminary characterization of native and selenomethionine-derivative crystals is reported. The Picornaviridae family contains a large number of human pathogens such as rhinovirus, poliovirus and hepatitis A virus (HAV). Hepatitis A is an infectious disease that causes liver inflammation. It is highly endemic in developing countries with poor sanitation, where infections often occur in children. As in other picornaviruses, the genome of HAV contains one open reading frame encoding a single polyprotein that is subsequently processed by viral proteinases to originate mature viral proteins during and after the translation process. In the polyprotein, the N-terminal P1 region generates the four capsid proteins, while the C-terminal P2 and P3 regions contain the enzymes, precursors and accessory proteins essential for polyprotein processing and virus replication. Here, the first crystals of protein 2AB of HAV are reported. The crystals belonged to space group P4 1 or P4 3 , with unit-cell parameters a = b = 90.42, c = 73.43 Å, and contained two molecules in the asymmetric unit. Native and selenomethionine-derivative crystals diffracted to 2.7 and 3.2 Å resolution, respectively

  15. Role of Matricellular Proteins in Disorders of the Central Nervous System.

    Science.gov (United States)

    Jayakumar, A R; Apeksha, A; Norenberg, M D

    2017-03-01

    Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell-cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell-cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down's syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson's disease and epilepsy. Potential therapeutic opportunities of MCP's for these disorders are also considered in this review.

  16. Construction and prokaryotic expression of the fusion gene PRRSV ...

    African Journals Online (AJOL)

    ajl4

    2013-07-24

    Jul 24, 2013 ... The fusion expressing plasmid pET32-GP5-Hsp70 was constructed and expressed in ... 2004). Hsps, expressed by prokaryotes and eukaryotes and their action as molecular ..... Facts, thoughts, and dreams. Shock. 12(4): ...

  17. Introduktion af polte i PRRSV-besætninger

    DEFF Research Database (Denmark)

    Hoelstad, Bonnie Edahl; Sonne Kristensen, Charlotte; Qvist Pawlowski, Mia

    karantæne. En optimal karantæne blev defineret som en stald, der ikke delte luftrum med øvrige staldafsnit, havde separat indgang og kørte alt ind/alt ud. På trods af at poltene ikke var viræmiske ved løbning, fører studiet ikke til ændringer i anbefalingerne vedrørende introduktion af polte i PRRS...

  18. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  19. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  20. Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

    Science.gov (United States)

    Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

    2015-04-19

    Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

  1. Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

    International Nuclear Information System (INIS)

    Tong, Xiaohang; Ma, Yanlin; Li, Xuemei

    2010-01-01

    The C-terminal domain of mouse hepatitis virus nucleocapsid protein has been overexpressed in E. coli, purified and crystallized. The crystal belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å, and diffracted to 2.20 Å resolution. Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (V M = 2.16 Å 3 Da −1 )

  2. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  3. Porcine arterivirus activates the NF-κB pathway through IκB degradation

    International Nuclear Information System (INIS)

    Lee, Sang-Myeong; Kleiboeker, Steven B.

    2005-01-01

    Nuclear factor-kappaB (NF-κB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-κB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-κB activation was characterized by translocation of NF-κB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-κB-regulated gene expression. NF-κB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-κB activation. Degradation of IκB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IκBα (IκBαDN) significantly suppressed NF-κB activation induced by PRRSV. However, IκBαDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-κB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-κB was activated by PRRSV infection. Moreover, NF-κB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-κB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV

  4. RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

    Science.gov (United States)

    Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

    2018-04-15

    Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral

  5. A thiophene-modified screen printed electrode for detection of dengue virus NS1 protein.

    Science.gov (United States)

    Silva, M M S; Dias, A C M S; Cordeiro, M T; Marques, E; Goulart, M O F; Dutra, R F

    2014-10-01

    A thiophene-modified screen printed electrode (SPE) for detection of the Dengue virus non-structural protein 1 (NS1), an important marker for acute phase diagnosis, is described. A sulfur-containing heterocyclic compound, the thiophene was incorporated to a carbon ink to prepare reproducible screen printed electrodes. After cured, the thiophene SPE was coated by gold nanoparticles conjugated to Protein A to form a nanostrutured surface. The Anti-NS1 antibodies immobilized via their Fc portions via Protein A, leaving their antigen specific sites free circumventing the problem of a random antibodies immobilization. Amperometric responses to the NS1 protein of dengue virus were obtained by cyclic voltammetries performed in presence of ferrocyanide/ferricyanide as redox probe. The calibration curve of immunosensor showed a linear response from 0.04 µg mL(-1) to 0.6 µg mL(-1) of NS1 with a good linear correlation (r=0.991, pink enhanced the electroanalytical properties of the SPEs, increasing their reproducibility and sensitivity. This point-of-care testing represents a great potential for use in epidemic situations, facilitating the early diagnosis in acute phase of dengue virus. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV Using Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Aurore Chevin

    2015-06-01

    Full Text Available Chronic bee paralysis virus (CBPV is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt and RNA 2 (2305 nt encode three and four putative open reading frames (ORFs, respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.

  7. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  8. Mapping of nuclear import signal and importin α3 binding regions of 52K protein of bovine adenovirus-3

    International Nuclear Information System (INIS)

    Paterson, Carolyn P.; Ayalew, Lisanework E.; Tikoo, Suresh K.

    2012-01-01

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ( 105 RKR 107 ) of the identified domain (amino acids 102 GMPRKRVLT 110 ) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin α/β-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin α3. Although deletion of amino acid 102–110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90–133 are required for interaction with importin-α3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin α3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  9. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

    Science.gov (United States)

    Suzich, J A; Tamura, J K; Palmer-Hill, F; Warrener, P; Grakoui, A; Rice, C M; Feinstone, S M; Collett, M S

    1993-01-01

    Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses. Images PMID:8396675

  10. The chaperone like function of the nonhistone protein HMGB1

    International Nuclear Information System (INIS)

    Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

    2013-01-01

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  11. What can we learn by computing 13Cα chemical shifts for X-ray protein models?

    International Nuclear Information System (INIS)

    Arnautova, Yelena A.; Vila, Jorge A.; Martin, Osvaldo A.; Scheraga, Harold A.

    2009-01-01

    The room-temperature X-ray structures of two proteins, solved at 1.8 and 1.9 Å resolution, are used to investigate whether a set of conformations, rather than a single X-ray structure, provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. The room-temperature X-ray structures of ubiquitin and of the RNA-binding domain of nonstructural protein 1 of influenza A virus solved at 1.8 and 1.9 Å resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and R free factors similar to those of the deposited X-ray structure, the 13 C α chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed 13 C α chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed 13 C α chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed 13 C α chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed 13 C α chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with

  12. Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection.

    Science.gov (United States)

    Du, Taofeng; Shi, Yunpeng; Xiao, Shuqi; Li, Na; Zhao, Qin; Zhang, Angke; Nan, Yuchen; Mu, Yang; Sun, Yani; Wu, Chunyan; Zhang, Hongtao; Zhou, En-Min

    2017-10-10

    Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

  13. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    International Nuclear Information System (INIS)

    Tzeng, W.-P.; Frey, Teryl K.

    2006-01-01

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles

  14. Membrane topology and cellular dynamics of foot-and-mouth disease virus 3A protein.

    Directory of Open Access Journals (Sweden)

    Mónica González-Magaldi

    Full Text Available Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP fused to the complete 3A (GFP3A and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.

  15. Comparing the intra-annual wood formation of three European species (Fagus sylvatica, Quercus petraea and Pinus sylvestris) as related to leaf phenology and non-structural carbohydrate dynamics.

    Science.gov (United States)

    Michelot, Alice; Simard, Sonia; Rathgeber, Cyrille; Dufrêne, Eric; Damesin, Claire

    2012-08-01

    Monitoring cambial phenology and intra-annual growth dynamics is a useful approach for characterizing the tree growth response to climate change. However, there have been few reports concerning intra-annual wood formation in lowland temperate forests with high time resolution, especially for the comparison between deciduous and coniferous species. The main objective of this study was to determine how the timing, duration and rate of radial growth change between species as related to leaf phenology and the dynamics of non-structural carbohydrates (NSC) under the same climatic conditions. We studied two deciduous species, Fagus sylvatica L. and Quercus petraea (Matt.) Liebl., and an evergreen conifer, Pinus sylvestris L. During the 2009 growing season, we weekly monitored (i) the stem radial increment using dendrometers, (ii) the xylem growth using microcoring and (iii) the leaf phenology from direct observations of the tree crowns. The NSC content was also measured in the eight last rings of the stem cores in April, June and August 2009. The leaf phenology, NSC storage and intra-annual growth were clearly different between species, highlighting their contrasting carbon allocation. Beech growth began just after budburst, with a maximal growth rate when the leaves were mature and variations in the NSC content were low. Thus, beech radial growth seemed highly dependent on leaf photosynthesis. For oak, earlywood quickly developed before budburst, which probably led to the starch decrease quantified in the stem from April to June. For pine, growth began before the needles unfolding and the lack of NSC decrease during the growing season suggested that the substrates for radial growth were new assimilates of the needles from the previous year. Only for oak, the pattern determined from the intra-annual growth measured using microcoring differed from the pattern determined from dendrometer data. For all species, the ring width was significantly influenced by growth duration

  16. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

    Science.gov (United States)

    Hedil, Marcio; Sterken, Mark G; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

  17. Genomic analysis and pathogenic characteristics of Type 2 porcine reproductive and respiratory syndrome virus nsp2 deletion strains isolated in Korea.

    Science.gov (United States)

    Choi, Hwan-Won; Nam, Eeuri; Lee, Yoo Jin; Noh, Yun-Hee; Lee, Seung-Chul; Yoon, In-Joong; Kim, Hyun-Soo; Kang, Shien-Young; Choi, Young-Ki; Lee, Changhee

    2014-06-04

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine virus that exhibits genetic and pathogenic heterogeneity among isolates. The present study was conducted to determine the complete genome sequence and pathogenicity of two Korean type 2 PRRSV nonstructural protein 2 (nsp2) deletion mutants, CA-2 and KNU-12-KJ4. The full-length genomes of CA-2 and KNU-12-KJ4 were determined to be 15,018 and 15,019 nucleotides in length, excluding the poly(A) tail, respectively, which were 393- or 392-nucleotide shorter than that of the type 2 NA prototype strain VR-2332 due to the presence of notable large deletions within the nsp2 gene. The genomes of CA-2 and KNU-12-KJ4 consisted of a 189- or 190-nucleotide 5' untranslated region (UTR), a 14,677-nucleotide protein-coding region, and a 151-nucleotide 3' UTR. Whole genome evaluation revealed that the nucleotide sequences of CA-2 and KNU-12-KJ4 are most similar to each other (10.7% sequence divergence), and then to the Korean strain CA-1 (11.3% sequence divergence) and the US strain MN184C (13.1% sequence divergence), respectively. To evaluate the in vitro immunity of nsp2 deletion variants, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM-pCD163 cells infected with each virus strain using quantitative real-time RT-PCR. Cytokine genes including IL-8, IL-10, and TNF-α, and chemokines such as MCP-1 and RANTES were found to be significantly elevated in nsp2 deletion virus-infected PAM cells. In contrast, expression of interferons (IFN-β, γ, and λ) and antiviral genes including ISG-15, -54, and -56 were unchanged or down-regulated in PAM cells infected with the nsp2 deletion mutants. Animal studies to assess the pathogenicity of nsp2 deletion PRRSVs demonstrated that both CA-2 and KNU-12-KJ4 strains notably produce weight loss in infected pigs. Furthermore, the nsp2 deletion mutants replicated well in pigs with significantly increased and prolonged

  18. Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

    International Nuclear Information System (INIS)

    Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

    2008-01-01

    Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

  19. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  20. Toscana virus induces interferon although its NSs protein reveals antagonistic activity.

    Science.gov (United States)

    Gori Savellini, Gianni; Weber, Friedemann; Terrosi, Chiara; Habjan, Matthias; Martorelli, Barbara; Cusi, Maria Grazia

    2011-01-01

    Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-β), although its non-structural protein (NSs) could inhibit the induction of IFN-β if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-β expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-β transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

  1. Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Leticia Barboza Rocha

    2017-10-01

    Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

  2. Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from MARC-145 cell line

    Directory of Open Access Journals (Sweden)

    Provost Chantale

    2012-11-01

    Full Text Available Abstract Background Airborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV, need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro. Results The SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFNα antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFNβ mRNA and no up regulation of IFNα mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFNγ and TNF-α mRNAs only in infected MARC-145 cells. Conclusions In conclusion, the SJPL cells are permissive to PRRSV. In addition, they are

  3. The C protein of measles virus inhibits the type I interferon response

    International Nuclear Information System (INIS)

    Shaffer, Jessica A.; Bellini, William J.; Rota, Paul A.

    2003-01-01

    Type I interferons (IFNα/β) are an important part of innate immunity to viral infections because they induce an antiviral response and limit viral replication until the adaptive response clears the infection. Since the nonstructural proteins of several paramyxoviruses inhibit the IFNα/β response, we chose to explore the role of the C protein of measles virus (MV) in such inhibition. Previous studies have suggested that the MV C protein may serve as a virulence factor, but its role in the pathogenesis of MV remains undefined. In the present study, a recombinant MV strain that does not express the C protein (MV C-) and its parental strain (Ed Tag) were used. Growth of MV C- was restricted in human peripheral blood mononuclear cells and HeLa cells, but in the presence of neutralizing antibodies to IFNα/β, MV C- produced titers that were equivalent to those of Ed Tag. In addition, expression of the MV C protein from plasmid DNA inhibited the production of an IFNα/β responsive reporter gene and, to a lesser extent, inhibited an IFNγ responsive reporter gene. The ability of the MV C protein to suppress the IFNα/β response was confirmed using a biologic assay. After IFNβ stimulation, HeLa cells infected with Ed Tag produced five-fold less IFNα/β than cells infected with MV C-. While the mechanism of inhibition remains unclear, these data suggest that the MV C protein plays an important role in the pathogenesis of MV by inhibiting IFNα/β signaling

  4. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  5. Determination of 5 '-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Nielsen, Jens

    1999-01-01

    We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length...... (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide...

  6. Progress toward an enhanced vaccine: Eight marked attenuated viruses to porcine reproductive and respiratory disease virus.

    Science.gov (United States)

    Spear, Allyn; Wang, Feng-Xue; Kappes, Matthew A; Das, Phani B; Faaberg, Kay S

    2018-03-01

    Recombinant viruses of strain Ingelvac® PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Δ23 and Δ87) and also the same deletions supplanted with foreign tags (Δ23-V5, Δ23-FLAG, Δ23-S, Δ87-V5, Δ87-FLAG, Δ87-S). The viruses, but one (Δ87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Δ23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Δ87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Δ23-S and Δ87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals. Published by Elsevier Inc.

  7. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-02-01

    Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  8. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

    Directory of Open Access Journals (Sweden)

    Tetsuro Ikegami

    2009-02-01

    Full Text Available Rift Valley fever virus (RVFV (genus Phlebovirus, family Bunyaviridae is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR-mediated eukaryotic initiation factor (eIF2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  9. Protein assay structured on paper by using lithography

    Science.gov (United States)

    Wilhelm, E.; Nargang, T. M.; Al Bitar, W.; Waterkotte, B.; Rapp, B. E.

    2015-03-01

    There are two main challenges in producing a robust, paper-based analytical device. The first one is to create a hydrophobic barrier which unlike the commonly used wax barriers does not break if the paper is bent. The second one is the creation of the (bio-)specific sensing layer. For this proteins have to be immobilized without diminishing their activity. We solve both problems using light-based fabrication methods that enable fast, efficient manufacturing of paper-based analytical devices. The first technique relies on silanization by which we create a flexible hydrophobic barrier made of dimethoxydimethylsilane. The second technique demonstrated within this paper uses photobleaching to immobilize proteins by means of maskless projection lithography. Both techniques have been tested on a classical lithography setup using printed toner masks and on a lithography system for maskless lithography. Using these setups we could demonstrate that the proposed manufacturing techniques can be carried out at low costs. The resolution of the paper-based analytical devices obtained with static masks was lower due to the lower mask resolution. Better results were obtained using advanced lithography equipment. By doing so we demonstrated, that our technique enables fabrication of effective hydrophobic boundary layers with a thickness of only 342 μm. Furthermore we showed that flourescine-5-biotin can be immobilized on the non-structured paper and be employed for the detection of streptavidinalkaline phosphatase. By carrying out this assay on a paper-based analytical device which had been structured using the silanization technique we proofed biological compatibility of the suggested patterning technique.

  10. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  11. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  12. Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I.

    Science.gov (United States)

    Gori-Savellini, Gianni; Valentini, Melissa; Cusi, Maria Grazia

    2013-06-01

    Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71-79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.

  13. Cellular Hsp27 interacts with classical swine fever virus NS5A protein and negatively regulates viral replication by the NF-κB signaling pathway.

    Science.gov (United States)

    Ling, Shifeng; Luo, Mingyang; Jiang, Shengnan; Liu, Jiayu; Ding, Chunying; Zhang, Qinghuan; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2018-05-01

    Classical swine fever virus (CSFV) nonstructural protein NS5A is a multifunctional protein functioning in regulation of viral genome replication, protein translation and assembly by interaction with viral or host proteins. Here, heat shock protein 27 (Hsp27) has been identified as a novel binding partner of NS5A by using His tag "pull down" coupled with shotgun LC-MS/MS, with interaction of both proteins further confirmed by co-immunoprecipitation and laser confocal assays. In PK-15 cells, silencing of Hsp27 expression by siRNA enhanced CSFV replication, and upregulation of Hsp27 inhibited viral proliferation. Additionally, we have shown that overexpression of Hsp27 increased NF-κB signaling induced by TNFα. Blocking NF-κB signaling in PK-15 cells overexpressing Hsp27 by ammonium pyrrolidinedithiocarbamate (PDTC) eliminated the inhibition of CSFV replication by Hsp27. These findings clearly demonstrate that the inhibition of CSFV replication by Hsp27 is mediated via the NF-κB signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, A.; Wong, S; Yuan, Y

    2009-01-01

    Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

  15. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  16. Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

    Directory of Open Access Journals (Sweden)

    Michael Breen

    Full Text Available Influenza A and B viruses (IAV and IBV, respectively cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1 was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

  17. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

    Science.gov (United States)

    Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

    2015-01-01

    Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

  18. Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

    Directory of Open Access Journals (Sweden)

    Pierre-Emmanuel Joubert

    2015-08-01

    Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

  19. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

    Science.gov (United States)

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

    2016-07-01

    Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs

  20. The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

    Science.gov (United States)

    Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

    2017-10-27

    The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    International Nuclear Information System (INIS)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-01-01

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication

  2. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  3. A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate.

    Science.gov (United States)

    Lauck, Michael; Sibley, Samuel D; Lara, James; Purdy, Michael A; Khudyakov, Yury; Hyeroba, David; Tumukunde, Alex; Weny, Geoffrey; Switzer, William M; Chapman, Colin A; Hughes, Austin L; Friedrich, Thomas C; O'Connor, David H; Goldberg, Tony L

    2013-08-01

    GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

  4. Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2006-12-01

    Full Text Available Abstract Background The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

  5. The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion.

    Directory of Open Access Journals (Sweden)

    Loïc Etienne

    Full Text Available Hepatitis C virus (HCV assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.

  6. Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

    Science.gov (United States)

    DeBlasio, Stacy L; Bereman, Michael S; Mahoney, Jaclyn; Thannhauser, Theodore W; Gray, Stewart M; MacCoss, Michael J; Cilia Heck, Michelle

    2017-09-01

    Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way to characterize virus-host interactomes in a single experiment. Common co-IP methods use antibodies immobilized on agarose or magnetic beads to isolate virus-host complexes in solutions of host tissue homogenate. Although these workflows are well established, they can be fairly laborious and expensive. Therefore, we evaluated the feasibility of using antibody-coated microtiter plates coupled with MS analysis as an easy, less expensive way to identify host proteins that interact with Potato leafroll virus (PLRV), an insect-borne RNA virus that infects potatoes. With the use of the bead-free platform, we were able to detect 36 plant and 1 nonstructural viral protein significantly coimmunoprecipitating with PLRV. Two of these proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2 (mMDH2), were detected as having a weakened or lost association with a structural mutant of the virus, demonstrating that the bead-free method is sensitive enough to detect quantitative differences that can be used to pin-point domains of interaction. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to identify plant and viral proteins interacting with virions and/or the viral structural proteins.

  7. The E5 Proteins

    OpenAIRE

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating ...

  8. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  9. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

    Science.gov (United States)

    Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

    2016-06-01

    glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes. Copyright © 2016 Varela et al.

  10. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  11. Protein docking prediction using predicted protein-protein interface.

    Science.gov (United States)

    Li, Bin; Kihara, Daisuke

    2012-01-10

    Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  12. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  13. Introduction to protein blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

    Protein blotting is a powerful and important procedure for the immunodetection of proteins following electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer.

  14. Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein.

    Science.gov (United States)

    Gargett, Tessa; Grubor-Bauk, Branka; Miller, Darren; Garrod, Tamsin; Yu, Stanley; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

    2014-06-01

    The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.

  15. In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

    Directory of Open Access Journals (Sweden)

    Jeremiah Athmer

    2017-01-01

    Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

  16. Matrix reloaded: CCN, tenascin and SIBLING group of matricellular proteins in orchestrating cancer hallmark capabilities.

    Science.gov (United States)

    Thakur, Ravi; Mishra, Durga Prasad

    2016-12-01

    Matricellular proteins (MCPs) are the non-structural extracellular matrix (ECM) proteins with various regulatory functions. MCPs are critical regulators of ECM homeostasis and are often found dysregulated in various malignancies. They interact with various proteins like ECM structural proteins, integrins, growth factor receptors and growth factors to modulate their availability and activity. Cancer-supporting MCPs are known to induce proliferation, migration and invasion of cancer cells. MCPs also support cancer stem (like) cell growth and induce a drug-resistant state. Apart from their direct effects on cancer cells, they play key roles in angiogenesis, immunomodulation, stromal cell infiltration, stromal proliferation and matrix remodeling. High expression of various MCPs belonging to the tenascin, CCN and SIBLING families is often associated with aggressive tumors and poor patient prognosis. Due to their differential expression and distinct functional role, these MCPs are perceived as attractive therapeutic targets in cancer. Studies on preclinical models have indicated that targeting tumor-supportive MCPs could be a potent avenue for developing anti-cancer therapies. The MCP receptors, like integrins and some associated growth factor receptors, are already being targeted using pharmacological inhibitors and neutralizing antibodies. Neutralizing antibodies against CCNs, tenascins and SIBLINGs have shown promising results in preclinical cancer models, suggesting an opportunity to develop anti-MCP therapies to target cancer. Peptides derived from anti-cancer MCPs could also serve as therapeutic entities. In the present review, in continuation with the expanding horizon of MCP functions and disease association, we focus on (i) their unique domain arrangement, (ii) their association with cancer hallmarks and (iii) available and possible therapeutic interventions. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

    Directory of Open Access Journals (Sweden)

    Tsubasa Munakata

    2007-09-01

    Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

  18. Silencing of the rotavirus NSP4 protein decreases the incidence of biliary atresia in murine model.

    Directory of Open Access Journals (Sweden)

    Jiexiong Feng

    Full Text Available Biliary atresia is a common disease in neonates which causes obstructive jaundice and progressive hepatic fibrosis. Our previous studies indicate that rotavirus infection is an initiator in the pathogenesis of experimental biliary atresia (BA through the induction of increased nuclear factor-kappaB and abnormal activation of the osteopontin inflammation pathway. In the setting of rotavirus infection, rotavirus nonstructural protein 4 (NSP4 serves as an important immunogen, viral protein 7 (VP7 is necessary in rotavirus maturity and viral protein 4 (VP4 is a virulence determiner. The purpose of the current study is to clarify the roles of NSP4, VP7 and VP4 in the pathogenesis of experimental BA. Primary cultured extrahepatic biliary epithelia were infected with Rotavirus (mmu18006. Small interfering RNA targeting NSP4, VP7 or VP4 was transfected before rotavirus infection both in vitro and in vivo. We analyzed the incidence of BA, morphological change, morphogenesis of viral particles and viral mRNA and protein expression. The in vitro experiments showed NSP4 silencing decreased the levels of VP7 and VP4, reduced viral particles and decreased cytopathic effect. NSP4-positive cells had strongly positive expression of integrin subunit α2. Silencing of VP7 or VP4 partially decreased epithelial injury. Animal experiments indicated after NSP4 silencing, mouse pups had lower incidence of BA than after VP7 or VP4 silencing. However, 33.3% of VP4-silenced pups (N = 6 suffered BA and 50% of pups (N = 6 suffered biliary injury after VP7 silencing. Hepatic injury was decreased after NSP4 or VP4 silencing. Neither VP4 nor VP7 were detected in the biliary ducts after NSP4. All together, NSP4 silencing down-regulates VP7 and VP4, resulting in decreased incidence of BA.

  19. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  20. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  1. Protein in diet

    Science.gov (United States)

    Diet - protein ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a ... to eat animal products to get all the protein you need in your diet. Amino acids are ...

  2. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  3. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  4. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

    Science.gov (United States)

    Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

    2010-05-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

  5. Nuclear relocalization of polyadenylate binding protein during rift valley fever virus infection involves expression of the NSs gene.

    Science.gov (United States)

    Copeland, Anna Maria; Altamura, Louis A; Van Deusen, Nicole M; Schmaljohn, Connie S

    2013-11-01

    Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.

  6. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

    Science.gov (United States)

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R; Kehn-Hall, Kylene; Omichinski, James G

    2015-05-12

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

  7. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    International Nuclear Information System (INIS)

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.; Borrego, Belen; Brocchi, Emiliana; Armas-Portela, Rosario; Sobrino, Francisco

    2008-01-01

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV (∼ 5 log) and VSV (∼ 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells

  8. High virulence differences among phylogenetically distinct isolates of the fish rhabdovirus viral hemorrhagic septicaemia virus are not explained by variability of the surface glycoprotein G or the non-virion protein Nv

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Harmache, Abdallah; Biacchesi, Stéphane

    2014-01-01

    -related novirhabdovirus [infectious hematopoietic necrosis virus (IHNV)], four chimaeric IHNV–VHSV recombinant viruses were generated. These chimaeric viruses included substitution of the IHNV glyco- (G) or non-structural (Nv) protein with their counterparts from either a trout-derived or a marine VHSV strain....... Comparative challenge experiments in rainbow trout fingerlings revealed similar levels of survival induced by the recombinant (r)IHNV–VHSV chimaeric viruses regardless of whether the G or Nv genes originated from VHSV isolated from a marine fish species or from rainbow trout. Interestingly, recombinant IHNV...... gained higher virulence following substitution of the G gene with those of the VHSV strains, whilst the opposite was the case following substitution of the Nv genes....

  9. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation.

    Science.gov (United States)

    Das, Lalatendu Keshary; Kundu, S S; Kumar, Dinesh; Datt, Chander

    2015-02-01

    To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO) and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS). Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier) were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash), fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose), primary CHO fractions (CHO, non-structural CHO, structural CHO and starch) and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein). The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - non-degrading or unavailable) and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable) fractions of test feeds. Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Based on our findings, it was concluded that feeds with similar CP and CHO content

  10. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation

    Directory of Open Access Journals (Sweden)

    Lalatendu Keshary Das

    2015-02-01

    Full Text Available Aim: To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS. Materials and Methods: Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash, fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose, primary CHO fractions (CHO, non-structural CHO, structural CHO and starch and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein. The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - nondegrading or unavailable and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable fractions of test feeds. Results: Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Conclusion: Based on our findings, it

  11. Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

    Science.gov (United States)

    2012-01-01

    Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

  12. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  13. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  14. Protein Structure Prediction by Protein Threading

    Science.gov (United States)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  15. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  16. Amino acids and proteins

    Science.gov (United States)

    A balanced, safe diet with proteins is important to meet nutritional requirements. Proteins occur in animal as well as vegetable products in important quantities. In some countries, many people obtain much of their protein from animal products. In other regions, the major portion of dietary protein ...

  17. Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

    Science.gov (United States)

    Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

    2012-11-30

    Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

    International Nuclear Information System (INIS)

    Vende, Patrice; Tortorici, M. Alejandra; Taraporewala, Zenobia F.; Patton, John T.

    2003-01-01

    The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates

  19. The Protein Model Portal

    OpenAIRE

    Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

    2008-01-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

  20. A proteomic perspective of inbuilt viral protein regulation: pUL46 tegument protein is targeted for degradation by ICP0 during herpes simplex virus type 1 infection.

    Science.gov (United States)

    Lin, Aaron E; Greco, Todd M; Döhner, Katinka; Sodeik, Beate; Cristea, Ileana M

    2013-11-01

    Much like the host cells they infect, viruses must also regulate their life cycles. Herpes simples virus type 1 (HSV-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. Following entry into host cells, the virion-associated outer tegument proteins pUL46 and pUL47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. Because pUL46 has gone largely unstudied, we employed a hybrid mass spectrometry-based approach to determine how pUL46 exerts its functions during early stages of infection. For a spatio-temporal characterization of pUL46, time-lapse microscopy was performed in live cells to define its dynamic localization from 2 to 24 h postinfection. Next, pUL46-containing protein complexes were immunoaffinity purified during infection of human fibroblasts and analyzed by mass spectrometry to investigate virus-virus and virus-host interactions, as well as post-translational modifications. We demonstrated that pUL46 is heavily phosphorylated in at least 23 sites. One phosphorylation site matched the consensus 14-3-3 phospho-binding motif, consistent with our identification of 14-3-3 proteins and host and viral kinases as specific pUL46 interactions. Moreover, we determined that pUL46 specifically interacts with the viral E3 ubiquitin ligase ICP0. We demonstrated that pUL46 is partially degraded in a proteasome-mediated manner during infection, and that the catalytic activity of ICP0 is responsible for this degradation. This is the first evidence of a viral protein being targeted for degradation by another viral protein during HSV-1 infection. Together, these data indicate that pUL46 levels are tightly controlled and important for the temporal regulation of viral gene expression throughout the virus life cycle. The concept of a structural virion protein, pUL46, performing nonstructural roles is likely to reflect a theme common to many viruses

  1. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  2. Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome do not change the cellular tropism of equine arteritis virus

    Science.gov (United States)

    Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they share many biological properties but differ significantly in cellular tropism. Using an infectious cDNA clone of EAV, we engineered a panel of six chimeric viruses b...

  3. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

    Science.gov (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

    2018-05-01

    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  4. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

  5. Personalizing Protein Nourishment