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Find video protocols related to scientific articles indexed in Pubmed.
Quantitative Proteomic Analysis Reveals That Transmissible Gastroenteritis Virus Activates the JAK-STAT1 Signaling Pathway.
J. Proteome Res.
PUBLISHED: 10-31-2014
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Transmissible gastroenteritis virus (TGEV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea and severe dehydration in piglets. In this study, liquid chromatography-tandem mass spectrometry coupled to isobaric tags for relative and absolute quantification labeling was used to quantitatively identify differentially expressed cellular proteins after TGEV infection in PK-15 cells. In total, 162 differentially expressed cellular proteins were identified, including 60 upregulated proteins and 102 downregulated proteins. These differentially expressed proteins were involved in the cell cycle, cellular growth and proliferation, the innate immune response, etc. Interestingly, many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1 (STAT1) and interferon-stimulated genes (ISGs). Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression. This study for the first time reveals that TGEV induces interferon signaling from the point of proteomic analysis.
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A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-22-2014
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The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-?B (NF-?B) and the NF-?B-regulated genes encoding tumor necrosis factor-? (TNF-?) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.
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Porcine reproductive and respiratory syndrome virus infection triggers HMGB1 release to promote inflammatory cytokine production.
Virology
PUBLISHED: 08-14-2014
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The high mobility group box 1 (HMGB1) protein is an endogenous damage-associated molecular pattern (DAMP) molecule involved in the pathogenesis of various infectious agents. Based on meta-analysis of all publicly available microarray datasets, HMGB1 has recently been proposed as the most significant immune modulator during the porcine response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, the function of HMGB1 in PRRSV pathogenesis is unclear. In this study, we found that PRRSV infection triggers the translocation of HMGB1 from the nucleus to the extracellular milieu in MARC-145 cells and porcine alveolar macrophages. Although HMGB1 has no effect on PRRSV replication, HMGB1 promotes PRRSV-induced NF-?B activation and subsequent expression of inflammatory cytokines through receptors RAGE, TLR2 and TLR4. Our findings show that HMGB1 release, triggered by PRRSV infection, enhances the efficiency of virus-induced inflammatory responses, thereby providing new insights into the pathogenesis of PRRSV infection.
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Hepatitis A virus 3C protease cleaves NEMO to impair induction of beta interferon.
J. Virol.
PUBLISHED: 06-11-2014
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NEMO (NF-?B essential modulator) is a bridging adaptor indispensable for viral activation of interferon (IFN) antiviral response. Herein, we show that hepatitis A virus (HAV) 3C protease (3Cpro) cleaves NEMO at the Q304 residue, negating its signaling adaptor function and abrogating viral induction of IFN-? synthesis via the retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5) and Toll-like receptor 3 (TLR3) pathways. NEMO cleavage and IFN antagonism, however, were lost upon ablation of the catalytic activity of 3Cpro. These data describe a novel immune evasion mechanism of HAV.
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Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1.
J. Virol.
PUBLISHED: 05-28-2014
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Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets and results in large economic losses in many Asian and European countries. A large-scale outbreak of porcine epidemic diarrhea occurred in China in 2010, and the virus emerged in the United States in 2013 and spread rapidly, posing significant economic and public health concerns. Previous studies have shown that PEDV infection inhibits the synthesis of type I interferon (IFN), and viral papain-like protease 2 has been identified as an IFN antagonist. In this study, we found that the PEDV-encoded nucleocapsid (N) protein also inhibits Sendai virus-induced IFN-? production, IFN-stimulated gene expression, and activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-?B. We also found that N protein significantly impedes the activation of the IFN-? promoter stimulated by TBK1 or its upstream molecules (RIG-I, MDA5, IPS-1, and TRAF3) but does not counteract its activation by IRF3. A detailed analysis revealed that the PEDV N protein targets TBK1 by direct interaction and that this binding sequesters the association between TBK1 and IRF3, which in turn inhibits both IRF3 activation and type I IFN production. Together, our findings demonstrate a new mechanism evolved by PEDV to circumvent the host's antiviral immunity.
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Molecular cloning and functional characterization of porcine DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41).
Dev. Comp. Immunol.
PUBLISHED: 04-25-2014
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DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), a member of the DEXDc helicase family, was recently identified as an intracellular DNA sensor in mouse myeloid dendritic cells. In this study, porcine DDX41 (poDDX41) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated in porcine kidney (PK-15) cells. Full-length poDDX41 cDNA encodes 622 amino acid residues and contains a DEADc domain and a HELICc domain. poDDX41 mRNA is widely expressed in different tissues, especially the stomach and liver. Overexpression of poDDX41 in PK-15 cells induced IFN-? by activating transcription factors IRF3 and NF-?B. Knockdown of poDDX41 with siRNA significantly reduced IFN-? expression induced by poly(dA:dT), a double-stranded DNA (dsDNA) analogue, or pseudorabies virus, a dsDNA swine virus. Therefore, poDDX41 is involved in the dsDNA- and dsDNA-virus-mediated type I IFN signaling pathway in porcine kidney cells.
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Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.
Vet. Microbiol.
PUBLISHED: 03-23-2014
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Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus.
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Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.
J. Proteome Res.
PUBLISHED: 02-24-2014
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Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-?B signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection.
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Porcine reproductive and respiratory syndrome virus induces IL-1? production depending on TLR4/MyD88 pathway and NLRP3 inflammasome in primary porcine alveolar macrophages.
Mediators Inflamm.
PUBLISHED: 02-10-2014
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Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Previous studies have reported that PRRSV infection induced the production of IL-1 ? . However, the cellular sensors and signaling pathways involved in this process have not been elucidated yet. Here, we studied the mechanisms responsible for the production of IL-1 ? in response to highly pathogenic PRRSV. Upon PRRSV infection of primary porcine alveolar macrophages, both mRNA expression and secretion of IL-1 ? were significantly increased in a time- and dose-dependent manner. We also investigated the role of several pattern-recognition receptors and adaptor molecules in this response and showed that the TLR4/MyD88 pathway and its downstream signaling molecules, NF- ? B, ERK1/2, and p38 MAPKs, were involved in IL-1 ? production during PRRSV infection. Treatment with specific inhibitors or siRNA knockdown assays demonstrated that components of the NLRP3 inflammasome were crucial for IL-1 ? secretion but not for IL-1 ? mRNA expression. Furthermore, TLR4/MyD88/NF- ? B signaling pathway was involved in PRRSV-induced expression of NLRP3 inflammasome components. Together, our results deciphered the pathways leading from recognition of PRRSV to the production and release of IL-1 ? , providing a deeper knowledge of the mechanisms of PRRSV-induced inflammation responses.
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Porcine reproductive and respiratory syndrome virus infection activates NOD2-RIP2 signal pathway in MARC-145 cells.
Virology
PUBLISHED: 01-23-2014
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Nucleotide-binding oligomerization domains (NOD)-like receptors (NLRs) evolve as a group of germline-encoded receptors that detect cytosolic pathogen-associated molecular patterns. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide. By examining the expression kinetics of ten selected NLRs, NOD2 and NLRP3 were found to be continuously up-regulated in PRRSV-infected MARC-145 cells during 48 h of post-infection. Further study revealed that PRRSV infection enhanced the expression and phosphorylation of RIP2. Knockdown of NOD2 and RIP2 by siRNA significantly decreased PRRSV-induced phosphorylation of NF-?B subunit p65, JNK, Erk and p38 MAPK, as well as the expression of IL-6, IL-8, TNF-?, and RANTES in MARC-145 cells. Moreover, increased expression of NOD2 and RIP2 mRNA were observed in alveolar macrophages isolated from PRRSV-challenged piglets at 3, 7 and 10 day post-challenge. Collectively, our results revealed that PRRSV infection activates NOD2-RIP2 signaling pathway to induce pro-inflammatory response.
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A MYB coiled-coil transcription factor interacts with NSP2 and is involved in nodulation in Lotus japonicus.
New Phytol.
PUBLISHED: 01-10-2014
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Transcription factor complex formation is a central step in regulating gene expression. In this report, a novel MYB coiled-coil transcription factor referred to as IPN2, for Interacting Protein of NSP2, is described. The interaction between IPN2 and NSP2 was examined by protein pull-down assays and bimolecular fluorescence complementation (BiFC). Subcellular localization of proteins, gene expression and gene function were assessed in transgenic hairy roots expressing tagged recombinant proteins, promoter-reporter and RNA interference (RNAi) constructs, respectively. The GRAS domain of NSP2 and the coiled-coil domain of IPN2 were found to be responsible for the interaction between the two proteins. IPN2 had strong transcription activation activity, bound directly to the NIN gene promoter, and was localized to the nuclei of Lotus japonicus root cells. The expression of IPN2 was elevated during nodule development, coinciding with increased NSP2 gene expression during nodule organogenesis. RNAi-mediated knockdown expression of IPN2 did not affect arbuscular mycorrhizal development, but had deleterious effects on rhizobial infection and nodule formation in L. japonicus. These results demonstrate an important role of IPN2 in nodule organogenesis and place a new MYB transcription factor in the Nod signaling pathway.
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Molecular cloning, functional characterization and antiviral activity of porcine DDX3X.
Biochem. Biophys. Res. Commun.
PUBLISHED: 12-16-2013
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Human DDX3X is a newly discovered DEAD-box RNA helicase. In addition to involvement of eukaryotic gene expression regulation, human DDX3X has recently been demonstrated to be a critical molecule in innate immune signaling pathways and to contribute to type I interferon (IFN) induction. In the present study, porcine DDX3X was cloned by RT-PCR from PK-15 cells and its function in regulating IFN-? was characterized. The putative porcine DDX3X ORF encodes 662 amino acids possessing several conserved motifs. Sequence alignments indicated that porcine DDX3X has high identity at the amino acid level to those of horse (96.7%), mouse (97.6%), cattle (98.5%), dog (98.6%) and human (98.9%). Ectopic expression of porcine DDX3X significantly activated IFN-? expression, whereas knockdown of porcine DDX3X inhibited dsRNA- or Sendai virus (SeV)-induced IFN-?. Furthermore, porcine DDX3X co-localized with IPS-1, TBK1 and IKK?, and enhanced IFN-? promoter activation induced by these molecules. We also investigated the role of porcine DDX3X during porcine reproductive and respiratory syndrome virus (PRRSV) infection and found that overexpression of DDX3X significantly inhibited PRRSV replication, indicating that DDX3X is a potential antiviral agent.
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Complete genome sequence of a novel deletion porcine reproductive and respiratory syndrome virus strain.
Genome Announc
PUBLISHED: 07-27-2013
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Porcine reproductive and respiratory syndrome virus HZ-31 strain is different from any other previously sequenced porcine reproductive and respiratory syndrome virus strains. It contains a 59-amino acid (aa) discontinuous deletion in aa 467 to 474, aa 498 to 519, and aa 533 to 561 of nsp2. Here, we report the complete genome sequence of this novel Chinese virulent PRRSV variant.
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Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain.
BMC Genomics
PUBLISHED: 01-31-2013
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Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses.
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Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3 activation.
Vet. Immunol. Immunopathol.
PUBLISHED: 01-22-2013
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Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.
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MiR-125b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the NF-?B pathway.
PLoS ONE
PUBLISHED: 01-02-2013
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Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. To investigate the impact of cellular microRNAs (miRNAs) on the replication of PRRSV, we screened 10 highly conserved miRNAs implicated in innate immunity or antiviral function and identified miR-125b as an inhibitor of PRRSV replication. Virus titer and western blot assays demonstrated that miR-125b reduced PRRSV replication and viral gene expression in a dose-dependent manner in both MARC-145 cell line and primary porcine alveolar macrophages. Mechanistically, miR-125b did not target the PRRSV genome. Rather, it inhibited activation of NF-?B, which we found to be required for PRRSV replication. PRRSV, in turn, down-regulated miR-125b expression post-infection to promote viral replication. Collectively, miR-125b is an antiviral host factor against PRRSV, but it is subject to manipulation by PRRSV. Our study reveals an example of manipulation of a cellular miRNA by an arterivirus to re-orchestrate host gene expression for viral propagation and sheds new light on targeting host factors to develop effective control measures for PRRS.
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Ubiquitin-specific proteases 25 negatively regulates virus-induced type I interferon signaling.
PLoS ONE
PUBLISHED: 01-01-2013
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Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-?, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-?B), as well as the phosphorylation of IRF3 and NF-?B subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-?. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.
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Foot-and-mouth disease virus (FMDV) leader proteinase negatively regulates the porcine interferon-?1 pathway.
Mol. Immunol.
PUBLISHED: 08-11-2011
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Foot-and-mouth disease is a highly contagious viral disease caused by foot-and-mouth disease virus (FMDV) of wild and domestic cloven-hoofed animals, and causes an economically important disease in the swine industry. In this study, we found that the replication of FMDV in IBRS-2 cells could be significantly inhibited after treatment with the purified recombinant porcine interferon lambda 1 (IFN-?1), a newly identified type III interferon. However, FMDV could not activate the IFN-?1 promoter and IFN-?1 mRNA expression in infected IBRS-2 cells, suggesting that FMDV has evolved mechanisms to interrupt the antiviral function of IFN-?1. The cause of this inhibition was determined by screening all structural and non-structural proteins of FMDV, and the leader proteinase (L(pro)) was found to exhibit the highest potential to inhibit poly(I:C)-induced IFN-?1 promoter activity. Further study revealed that the catalytic activity and a SAP (SAF-A/B, Acinus, and PIAS) domain of L(pro) were required for suppressing poly(I:C)-induced IFN-?1 production. These data suggest that FMDV replication could be inhibited by porcine IFN-?1, but that the virus has evolved specific mechanisms to inhibit this action.
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Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine.
Antiviral Res.
PUBLISHED: 08-03-2011
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The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in veterinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5×10(7) IFU BV-HAs developed significantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% protection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses.
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Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication.
Virus Res.
PUBLISHED: 07-25-2011
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Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway that acts in the maintenance of cellular homeostasis and plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), an agent that has caused devastating losses in the international swine industry since the late 1980s. Using protein quantification and microscopy, we observed that PRRSV infection results in LC3-I/II conversion, an increased accumulation of punctate GFP-LC3-expressing cells, and a higher number of autophagosome-like double-membrane vesicles in the cytoplasm of host cells. Inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNAs targeting ATG7 and Beclin-1 led to a significant reduction in PRRSV titers and protein expression. Conversely, induction of autophagy by rapamycin resulted in increased viral replication. These results demonstrate that PRRSV infection induces autophagy which, in turn, enhances viral replication efficiency.
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Epidemiology and evolutionary characteristics of the porcine reproductive and respiratory syndrome virus in China between 2006 and 2010.
J. Clin. Microbiol.
PUBLISHED: 07-20-2011
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In 2006, an emerging highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, broke out in mainland China and spread rapidly to neighboring countries. To examine the epidemiology and evolutionary characteristics of Chinese PRRSV after the 2006 outbreak, we tested 2,981 clinical samples collected from 2006 to 2010 in China, determined 153 Nsp2 sequences and 249 ORF5 sequences, and analyzed the epidemiology and genetic diversity of Chinese PRRSV. Our results showed that the percentage of PRRSV-positive specimens collected from sick pigs averaged 60.85% in the past 5 years and that the highly pathogenic PRRSV has become the dominant strain in China. Furthermore, a reemerging strain which apparently evolved from the highly pathogenic PRRSV strain in 2006 appeared to be widely prevalent in China from 2009 onwards. Sequence analyses revealed that the hypervariable region of Nsp2 in most of the isolates contained a discontinuous deletion equivalent to 30 amino acids, along with other types of deletions. Extensive amino acid substitutions in the GP5 sequence translated from ORF5 were found, particularly in the potential neutralization epitope and the N-glycosylation sites. Our results suggest that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines. Information from this study will help in understanding the evolutionary characteristics of Chinese PRRSV and assist ongoing efforts to develop and use PRRSV vaccines in the future.
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Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells.
Sci China Life Sci
PUBLISHED: 05-27-2011
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Cholesterol represents one of the key constituents of small, dynamic, sterol- and sphingolipid-enriched domains on the plasma membrane. It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection. In this study, the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus (PRRSV) infection of MARC-145 cells was investigated. Pretreatment of MARC-145 cells with methyl-?-cyclodextrin (M?CD), a drug used to deplete cholesterol from cellular membrane, significantly reduced PRRSV infection in a dose-dependent manner. This inhibition was partially reversed by supplementing exogenous cholesterol following M?CD treatment, suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol. Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry, especially virus attachment and release. These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.
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Understanding Streptococcus suis serotype 2 infection in pigs through a transcriptional approach.
BMC Genomics
PUBLISHED: 05-20-2011
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Streptococcus suis serotype 2 (S. suis 2) is an important pathogen of pigs. S suis 2 infections have high mortality rates and are characterized by meningitis, septicemia and pneumonia. S. suis 2 is also an emerging zoonotic agent and can infect humans that are exposed to pigs or their by-products. To increase our knowledge of the pathogenesis of meningitis, septicemia and pneumonia in pigs caused by S. suis 2, we profiled the response of peripheral blood mononuclear cells (PBMC), brain and lung tissues to infection with S. suis 2 strain SC19 using the Affymetrix Porcine Genome Array.
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Antiviral activity of type I and type III interferons against porcine reproductive and respiratory syndrome virus (PRRSV).
Antiviral Res.
PUBLISHED: 04-22-2011
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The newly identified type III interferons (IFNs), also known as IFN-?1/IL-29, IFN-?2/IL-28A and IFN-?3/IL-28B, like type I IFNs, have antiviral activity against a broad spectrum of viruses. We therefore examined whether type III IFNs, as well as type I IFNs, has the ability to inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in MARC-145 cells. We found that replication of PRRSV in MARC-145 cells was significantly reduced following treatment with IFN-?1, IFN-?2 and IFN-?3, respectively, and such inhibition was dose-dependent. However, type III IFNs (IFN-?1, IFN-?2 and IFN-?3) was less effective than type I IFNs (IFN-? and IFN-?) in antiviral activity against PRRSV. Mixture of two types of IFNs could not improve the antiviral activity of each type alone. In addition, all types of IFNs in our study were able to induce the expression of ISG56, 2,5-OAS and MxA in MARC-145 cells. These data demonstrate that type III IFNs had antiviral activity against PRRSV and may serve as useful antiviral agents against infectious swine diseases.
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Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced RANTES transcription in PK-15 cells.
Virus Genes
PUBLISHED: 02-19-2011
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The chemokine RANTES (regulated upon activation, normal T-cells expressed and secreted) plays an essential role in inflammation and immune response. Infection with wild-type foot-and-mouth disease virus (FMDV) in PK-15 cells strongly inhibits the expression of RANTES compared to infection with a genetically engineered mutant lacking the leader protein (L(pro)) coding region. This suggests that L(pro) is involved in RANTES regulation. However, the underlying molecular mechanism remains unclear. In this study, we show that transfection of PK-15 cells with a plasmid expressing the L(pro) of FMDV, in the absence of other FMDV proteins, inhibited dsRNA-induced RANTES transcription and promoter activity. Promoter mutagenesis experiments revealed that the interferon-stimulated response element (ISRE) was important for the ability of L(pro) to inhibit dsRNA-induced RANTES promoter activity. Furthermore, over-expression of L(pro) also inhibited IRF-3/7-mediated RANTES activation. Screening L(pro) mutants indicated that catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of L(pro) were required to suppress dsRNA-induced RANTES transcription.
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The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase.
J. Virol.
PUBLISHED: 02-09-2011
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The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that L(pro) is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lb(pro) that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lb(pro) as well as its ability to block signaling to the IFN-? promoter. Collectively, these results demonstrate that FMDV Lb(pro) possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.
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Development of a novel TaqMan-based real-time PCR assay for the detection of porcine boca-like virus (Pbo-likeV).
Virol. J.
PUBLISHED: 01-28-2011
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The recently discovered porcine boca-like virus (Pbo-likeV) is a member of the Parvoviridae family, genus Bocavirus, and it is potentially associated with swine disease. Several studies have associated Pbo-likeV with postweaning multisystemic wasting syndrome in pigs, but the full spectrum of clinical disease and the epidemiology of Pbo-likeV infection remain unclear. The availability of rapid and reliable molecular diagnostics would aid future studies of this novel virus. Thus, we developed a sensitive and specific TaqMan-based real-time PCR assay to target the Pbo-likeV NP1 gene. The assay reproducibly detected 20 copies of a recombinant DNA plasmid containing the NP1 gene, with a dynamic range of six orders of magnitude (10(2)-10(7) copies). The assay did not cross-react with other animal viruses. Clinical evaluation found that Pbo-likeV was present in Chinese swine herds at a frequency of 44.2% (114/258). Higher infection rates were found in diseased pigs (56.1%, 101/180) compared with healthy pigs (16.7%, 13/78) (P < 0.05). Our assay for the diagnosis and quantification of Pbo-likeV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of Pbo-likeV infection.
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Complete coding sequences and phylogenetic analysis of porcine bocavirus.
J. Gen. Virol.
PUBLISHED: 01-12-2011
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Here we report, for the first time, the nearly full-length genome sequence of porcine bocavirus (PBoV), a recently discovered parvovirus from pigs. Phylogenetic trees based on this genome sequence showed that PBoV belongs to the branch containing the genus Bocavirus, which comprises canine minute virus (CnMV), bovine parvovirus, gorilla bocavirus and human bocavirus (HBoV), and was most closely related to the group containing CnMV. PBoV was predicted to contain three potential ORFs encoding the non-structural protein NS1, the characteristic NP1 protein and the capsid protein VP1/VP2, with an organization similar to that of known bocaviruses. Interestingly, the NS1 gene of PBoV was more similar in length to the homogeneous gene found in HBoV than to those of other known bocaviruses. In addition, highly conserved unique splice-donor and -acceptor sites were identified in the NS1 gene of HBoV and PBoV.
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Construction and immunogenicity of a recombinant pseudotype baculovirus expressing the glycoprotein of rabies virus in mice.
Arch. Virol.
PUBLISHED: 01-09-2011
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A pseudotype baculovirus with the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope was used as a vector for the construction of recombinant baculovirus expressing the G protein of rabies virus (RABV) under the cytomegalovirus (CMV) promoter. The generated recombinant baculovirus (BV-G) efficiently expressed the RABV G proteins in mammalian cells. Intramuscular vaccination with BV-G (10(9) PFU/mouse) induced the production of RABV G-specific neutralizing antibodies and strong T cell responses in mice. Our data clearly indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccine against RABV infection.
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Complete genome sequence of Mycoplasma hyopneumoniae strain 168.
J. Bacteriol.
PUBLISHED: 12-10-2010
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Mycoplasma hyopneumoniae strain 168, a pathogenic strain prevalent in China, was isolated in 1974. Although this strain has been widespread for a long time, the genome sequence had not been determined. Here, we announce the complete genome sequence of M. hyopneumoniae strain 168.
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Microarray analyses of THP-1 cells infected with Streptococcus suis serotype 2.
Vet. Microbiol.
PUBLISHED: 09-30-2010
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Streptococcus suis serotype 2 (S. suis 2) is a pathogen responsible for several diseases in both pigs and humans. To gain more insight into the pathogenesis of this organism, an oligonucleotide (oligo)-based microarray was used to investigate gene expression changes in human monocytic cells (THP-1) in response to exposure to S. suis 2 strain SC19. A total of 328 differentially expressed genes were identified. These differentially expressed genes belonged to a variety of functional categories, including genes involved in apoptosis, immunity, signal transduction, chemokine production and the ubiquitin-proteasome system. Our findings can be of interest for future research.
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Molecular cloning of the porcine RANTES promoter: functional characterization of dsDNA/dsRNA response elements in PK-15 cells.
Dev. Comp. Immunol.
PUBLISHED: 09-26-2010
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The chemokine RANTES plays an essential role in inflammation and immune response. In this study, we cloned the nucleotide sequence of the 5-flanking region of the porcine RANTES (poRANTES) gene and characterized the regulatory elements that activate transcription. Analyses of a series of 5 deletion constructs demonstrated that a 266 bp region (-220/+46) that spanned the potential transcription start site of the poRANTES gene was sufficient to activate transcription in PK-15 cells. Furthermore, our results indicated that dsDNA/dsRNA significantly induced poRANTES promoter activity and expression of mRNA levels in a time- and dose-dependent manner. Promoter deletions and mutagenesis experiments indicated that an interferon-stimulated responsive element (ISRE) was critical for dsDNA/dsRNA-induced poRANTES transcription. In addition, porcine interferon regulatory factor 3 (IRF-3) and IRF-7 play important roles in dsDNA/dsRNA-induced poRANTES expression.
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Porcine reproductive and respiratory syndrome virus (PRRSV) infection activates chemokine RANTES in MARC-145 cells.
Mol. Immunol.
PUBLISHED: 09-20-2010
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RANTES (regulated upon activation, normally T-cell expressed and presumably secreted), a CC chemokine, plays an important role in the inflammatory response associated with viral infections. Previous studies have demonstrated that infection with porcine reproductive and respiratory syndrome virus (PRRSV) induces RANTES transcription in vitro and in vivo. However, the molecular mechanism remains unclear. In this study, real-time RT-PCR and promoter luciferase reporter assays showed that PRRSV infection significantly upregulates RANTES gene transcription in both a time- and dose-dependent manner and this induction requires viral replication in MARC-145 cells. Promoter mutagenesis experiments found that the nuclear factor (NF-?B) binding sites play an important role in PRRSV-induced RANTES transcription, while the interferon-stimulated responsive element (ISRE) site is not essential. PRRSV-induced RANTES transcription was dramatically inhibited by administration of a dominant-negative mutant of I?B kinase alpha (mI?B?), NF-?B inhibitor BAY11-7082 or ERK1/2 inhibitor U0126. In addition, the use of dominant-negative mutants of various adaptor molecules of the Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling pathways demonstrated that PRRSV upregulated RANTES transcription is dependent on myeloid differentiation primary response gene 88 (MyD88), TIR domain-containing adaptor inducing IFN-? (TRIF) and TNF receptor-associated factor 6 (TRAF6), indicating that the TLR signaling pathway is involved in PRRSV-induced RANTES activation.
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Complete genome sequence of Mycoplasma hyorhinis strain HUB-1.
J. Bacteriol.
PUBLISHED: 08-27-2010
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Mycoplasma hyorhinis is generally considered a swine pathogen yet is most commonly found infecting laboratory cell lines. An increasing body of evidence suggests that chronic infections with M. hyorhinis may cause oncogenic transformation. Here, we announce the complete genome sequence of M. hyorhinis strain HUB-1.
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Molecular cloning, expression and antiviral activity of porcine interleukin-29 (poIL-29).
Dev. Comp. Immunol.
PUBLISHED: 08-24-2010
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Human interleukin-29 (IL-29) is a recently discovered cytokine displaying antiviral activity against a broad spectrum of viruses, designated as interferon (IFN)-?1. We here report the molecular cloning, expression and antiviral activity of porcine IL-29 (poIL-29). The full-length poIL-29 cDNA sequence encoded 191 amino acids with a 19 amino acid signal peptide. Sequence alignments showed that poIL-29 had amino acid sequence similarity to wolf (76%), bear (75%), horse (75%), orangutan (73%), human (72%), cat (72%) and dog (70%) IL-29. The poIL-29 without signal anchor sequence was efficiently expressed as a 6 × HIS fusion protein in Escherichia coli and the antiviral activity of the purified recombinant protein was 1.8 × 10(3)U/mg protein. The purified recombinant poIL-29 also exhibited significant antiviral effects against porcine reproductive and respiratory syndrome virus and pseudorabies virus in a dose-dependent manner, suggesting that poIL-29 is a potential antiviral agent against swine infectious diseases.
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N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro.
Vet. Res. Commun.
PUBLISHED: 07-21-2010
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Nitric oxide (NO) was proposed to be an important molecule against some microorganisms. In this study, we investigated the inhibitory effect of NO on the infection by porcine reproductive and respiratory syndrome virus (PRRSV) in vitro and the role of NO in the defense against PRRSV. Our results indicated that exogenous NO did not inhibit PRRSV infection. Unexpectedly, N-acetylpenicillamine (NAP), a commonly used compound as negative control for NO-producing reagents, inhibited PRRSV replication. Thus, the inhibition effect of NAP on PRRSV replication was further explored. We found that the maximal inhibition effect of NAP on PRRSV replication was achieved upon treatment 1 h after virus infection and the virus yield was reduced by approximately 50 fold in the presence of 400 muM NAP. An obvious inhibitory effect on viral RNA and protein synthesis was also observed. However, the inhibitory effect was only achieved at early phase of virus infection. The normal virus yield could be restored upon the removal of NAP treatment. The inhibitory effect might be caused by sulfhydryl-reducing capacity and metal chelating properties of NAP. These studies suggested that (i) NO production or NO synthase (NOS) expression profiling may not be a reliable index for the immune response to PRRSV; (ii) NAP could inhibit the replication of PRRSV.
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Activation of NF-?B by nucleocapsid protein of the porcine reproductive and respiratory syndrome virus.
Virus Genes
PUBLISHED: 07-14-2010
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Nuclear factor kappa B (NF-?B) is a critical transcription factor in innate and adaptive immune response as well as cell proliferation and survival. Previous studies have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection activated NF-?B pathways through I?B degradation in MARC-145 cells and alveolar macrophages. To evaluate the mechanisms behind this, we investigated the role of PRRSV structural proteins in the regulation of NF-?B. In this study, we screened the structural proteins of PRRSV by NF-?B DNA-binding assay and luciferase activity assay and demonstrated that PRRSV nucleocapsid (N) protein could activate NF-?B in MARC-145 cells. Furthermore, we revealed that the region between aa 30 and 73 of N protein was essential for its function in the activation of NF-?B. These results presented here provide a basis for understanding molecular mechanism of PRRSV infection and inflammation response.
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Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels.
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-07-2010
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The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-beta (IFN-beta) antagonist that disrupts the integrity of transcription factor nuclear factor kappaB (NF-kappaB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-alpha1/beta expression caused by L(pro) was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-alpha/beta. Furthermore, overexpression of L(pro) significantly reduced the transcription of multiple IRF-responsive genes including 2,5-OAS, ISG54, IP-10, and RANTES. Screening L(pro) mutants indicated that the ability to process eIF-4G of L(pro) is not required for suppressing dsRNA-induced activation of the IFN-alpha1/beta promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-kappaB, L(pro) also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.
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Molecular cloning and functional characterization of porcine stimulator of interferon genes (STING).
Dev. Comp. Immunol.
PUBLISHED: 03-15-2010
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The stimulator of interferon genes (STING) of human/mouse has been identified recently as an adaptor that links virus-sensing receptors to interferon regulatory factor 3 (IRF3) activation. Here we report the cloning and characterization of porcine STING (poSTING). The full-length poSTING cDNA sequence encodes 378 amino acids and contains one endoplasmic reticulum (ER) retention motif, RAR. Phylogenetic analysis revealed that poSTING, together with bovine STING, is more closely related to the human than to mouse STING. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Enhanced green fluorescence protein (EGFP)-labeled poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and nuclear factor kappaB (NF-kappaB) transcription factors to induce IFN-beta production, while knockdown of poSTING significantly inhibited poly(I:C)- and poly(dAT:dAT)-induced IFN-beta promoter activation and IFN-beta mRNA production. Furthermore, the pseudorabies virus (PRV), a dsDNA virus, has been shown to activate the IFN-beta promoter in a poSTING-dependent way in porcine cell lines. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution.
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The genomic diversity of Chinese porcine reproductive and respiratory syndrome virus isolates from 1996 to 2009.
Vet. Microbiol.
PUBLISHED: 01-12-2010
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Since it was first reported in 1995, porcine reproductive and respiratory syndrome (PRRS) has become one of the most important swine diseases in China. A large number of field PRRSV strains have been isolated from different regions of China at different times, especially after a highly pathogenic PRRSV emerged in 2006. Previous studies based on ORF5 gene sequences revealed extensive genetic diversity among Chinese PRRSV isolates. To fully understand the extent of genetic diversity of PRRSV in China, we determined the genomic sequence of PRRSV WUH1, a highly pathogenic PRRSV isolated in late 2006. Based on the complete genomic sequences of strain WUH1 and 66 other field Chinese PRRSV strains isolated from 1996 to 2009, we further analyzed their genetic diversity. The results showed that all the tested Chinese PRRSV isolates belong to the North American genotype and can be clearly divided into four highly diverse subgenotypes. Furthermore, the analysis supported the concept that the highly pathogenic PRRSV in China emerged by gradual variation and evolution from the Chinese domestic virus. In addition, different deletions within Nsp2, deletion and potential antigenic drift within GP3, and point mutations within GP5, were extensively observed in Chinese PRRSV isolates. Interestingly, in addition to a unique discontinuous deletion of 30 aa in Nsp2, there was a 1 nucleotide deletion in both the 5UTR and 3UTR in nearly all highly pathogenic PRRSV isolates. These results will contribute to the elucidation of the evolutionary mechanisms of PRRSV in China.
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Recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses.
Emerging Infect. Dis.
PUBLISHED: 12-08-2009
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Em2007, a porcine reproductive and respiratory syndrome virus (PRRSV) variant with a unique 68 aa deletion in Nsp2, was recently isolated in China. Phylogenetic and molecular evolutionary analyses indicated that Em2007 is a natural recombinant between a vaccine strain of PRRSV and circulating virus. We also tested its pathogenicity in piglets.
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Efficient gene delivery into mammalian cells by recombinant baculovirus containing a hybrid cytomegalovirus promoter/Semliki Forest virus replicon.
J Gene Med
PUBLISHED: 09-17-2009
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Baculovirus, which is widely utilized as an excellent tool for the production of recombinant protein in insect cells, has recently emerged as a novel and attractive gene delivery vehicle for mammalian cells. Alphavirus, such as Semliki Forest virus (SFV), has also received considerable attention for use as expression vectors because of its self-replicating property. In the present study, we investigated the characterization of recombinant baculovirus incorporating a hybrid cytomegalovirus (CMV) promoter/SFV replicon.
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Molecular cloning and functional characterization of porcine DNA-dependent activator of IFN-regulatory factors (DAI).
Dev. Comp. Immunol.
PUBLISHED: 08-05-2009
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The DNA-dependent activator of IFN-regulatory factors (DAI) is a recently identified DNA sensor for intracellular DNA that triggers a signal for the production of type I IFN. Here we report the cloning and characterization of porcine DAI (poDAI). The full-length of poDAI encodes 439 amino acids, contains two N-terminal DNA-binding domains and shows similarity to mouse, rat, dog, monkey, human, horse and cattle counterparts ranging from 44% to 67%. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. Over-expression of poDAI activated transcription factors IRF3 and NF-kappaB and induced IFN-beta in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-beta. siRNA targeting poDAI significantly decreased poly(dAT:dAT)- or Pseudorabies virus (PRV)-induced IFN-beta activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system.
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Development of a vaccine vector based on a subgenomic replicon of porcine reproductive and respiratory syndrome virus.
J. Virol. Methods
PUBLISHED: 03-30-2009
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In this study, a DNA-launched subgenomic replicon of porcine reproductive and respiratory syndrome virus (PRRSV) was developed for use as a vaccine vector. This replicon plasmid contained a PRRSV subgenome without structural genes ORF2-ORF6, and was under the transcriptional control of the immediate-early promoter of cytomegalovirus (CMV). Using enhanced green fluorescent protein (EGFP) as a reporter gene, the DNA-launched subgenomic replicon of PRRSV, named pOK-Clone20-rep, could express heterologous genes in vitro. After direct inoculation of pOK-Clone20-rep, mice developed antibody responses that were specific for both the EGFP and the N gene in a dose-dependent manner. Furthermore, mice immunized with pOK-Clone20-rep at a dose of 100microg showed significantly enhanced levels of IFN-gamma compared with those inoculated with 100microg of pcD-EGFP, a conventional DNA vaccine that encodes EGFP. In summary, the results show that the DNA-launched subgenomic replicon of PRRSV could not only mediate foreign gene expression in vitro but also induced an immune response in vivo. Similarly, expression and immunogenicity of the N gene also strengthened the potential of the replicon to serve as a vaccine vector expressing multiple genes. It therefore provides a useful tool for vaccine development and the study of the transcription and replication of PRRSV.
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Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice.
J Gene Med
PUBLISHED: 03-18-2009
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Japanese encephalitis (JE) is a serious infection disease throughout southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a matter of high priority. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was described as an attractive gene-delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed recombinant pseudotype baculovirus encoding the Japanese encephalitis virus (JEV) envelope (E) protein and demonstrated that it could elicit high protective immunity in mice.
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Enhanced immune response and protection efficacy of a DNA vaccine constructed by linkage of the Mycobacterium tuberculosis Ag85B-encoding gene with the BVP22-encoding gene.
J. Med. Microbiol.
PUBLISHED: 03-11-2009
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Plasmid DNA vaccines have been widely explored for use in tuberculosis immunization but their immunogenicity needs improvement. In the present study, we incorporated the bovine herpesvirus 1 VP22 (BVP22)-encoding gene, which encodes a protein that demonstrates a capability for disseminating the expressed antigen to neighbouring cells, into a DNA vector in which it was fused to the Ag85B-encoding gene of Mycobacterium tuberculosis (Mtb), and investigated whether this linkage could enhance immune response and protective efficacy in C57BL/6 mice compared to plasmid DNA encoding Ag85B alone. After immunization in mice, Ag85B-specific ELISA antibodies and spleen lymphocyte proliferative responses induced by DNA co-expressing BVP22 and Ag85B were significantly higher than those obtained in mice immunized with Ag85B-encoding DNA alone, except for the number of gamma interferon secreting cells. In addition, based on histopathological examination and bacterial-load determination in lung and spleen, protection against intravenous Mtb H37Rv challenge evoked by the BVP22-Ag85B DNA immunization exceeded the response elicited by Ag85B DNA alone, which was not significantly different from that provided by Bacillus Calmette-Guérin (BCG). These results suggested that DNA vaccine consisting of BVP22 and Ag85B-encoding DNA enhanced immune response and protection against intravenous Mtb H37Rv challenge in mice, indicating that BVP22-encoding DNA might be a promising tool to enhance TB DNA vaccine efficacy.
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A pseudotype baculovirus-mediated vaccine confers protective immunity against lethal challenge with H5N1 avian influenza virus in mice and chickens.
Mol. Immunol.
PUBLISHED: 02-24-2009
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Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein was used as a vector to express the hemagglutinin (HA) protein of highly pathogenic H5N1 avian influenza virus, A/Chicken/Hubei/327/2004 (HB/327). The resultant recombinant baculovirus (BV-G-HA) mediated gene delivery and HA expression efficiently in mammalian cells. Mice immunized with 1 x 10(9)PFU of BV-G-HA developed significantly higher levels of H5-specific antibodies and cellular immunity than those that received 100 microg of DNA vaccines expressing HA, and were completely protected from lethal challenge with HB/327. Different vaccination doses were further tested in chickens, and these experiments demonstrated that 1 x 10(8)PFU of BV-G-HA offered complete protection from challenge with 100 LD(50) of HB/327. These data indicate that the pseudotype baculovirus-mediated vaccine could be utilized as an alternative strategy against the pandemic spread of H5N1 influenza virus.
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Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice.
J Gene Med
PUBLISHED: 02-06-2009
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Japanese encephalitis (JE) is a serious infection and disease in southern and eastern Asia. The design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a high-priority target in the world. Recently, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) was described as an attractive gene delivery vehicle in mammalian cells and a potential vector for vaccine development. In the present study, we constructed a recombinant pseudotype baculovirus encoding the JEV envelope (E) protein and demonstrated that it could elicit high protective immunity in mice.
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Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene.
Vaccine
PUBLISHED: 01-12-2009
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Since May 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. The inability to induce robust protective immunity has been postulated to be associated with the presence of a non-neutralizing decoy epitope and heavy glycosylation in close to its neutralizing epitope. In this study, a synthetic ORF5 gene (SynORF5) was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of highly pathogenic PRRSV strain WUH3. Additional modifications, i.e., inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the synthetic ORF5 gene. The immunogenicity of the SynORF5-encoded GP5 was evaluated by DNA vaccination in mice and piglets. Results showed that significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-gamma level, as well as lymphocyte proliferation response, could be induced in mice and piglets immunized with DNA construct encoding the modified GP5 than those received DNA vaccine expressing the native GP5. The enhanced immunogenicity of the modified GP5 will be useful to facilitate the development of efficient vaccines against the highly pathogenic PRRSV in the future.
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Transcriptional suppression of IE180 and TK promoters by the EP0 of pseudorabies virus strains Ea and Fa.
Virus Genes
PUBLISHED: 01-07-2009
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In our transient expression assays, the IE180 and TK promoters were markedly suppressed by the EP0 of the pseudorabies virus strains Ea and Fa (EP0/Ea, EP0/Fa). This is in contrast with the transactivating activity of EP0 from strain YS-81 (EP0/YS-81) in previous studies. Amino acid sequence alignment revealed several mutations in both EP0/Ea and EP0/Fa compared with EP0/YS-81. Most remarkable is a two-amino acid substitution next to the RING finger domain which was considered to be important for the transactivating activity of EP0 in previous studies. To address the effect of the two-amino acid substitution on the function of EP0, reverse mutants of EP0/Ea and EP0/Fa were generated. The subsequent expression assays indicated that this substitution was at least in part responsible for the effect on the regulatory activity of EP0. Our data suggested that EP0 may regulate the expression of the same genes in different pseudorabies virus strains differently.
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Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-06-2009
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Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 microg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.
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A suicidal DNA vaccine co-expressing two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus antigens induce protective responses.
Biotechnol. Lett.
PUBLISHED: 01-01-2009
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We constructed a suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing GP5m (a modified GP5) and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV). In mice immunization, specific immune responses were elicited by the suicidal DNA vaccine pSFV-ORF5m/ORF6. The immunogenicity and protective efficiency was then evaluated in piglets immunized with pSFV-ORF5m/ORF6 before virus challenge: PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses were developed. Post-PRRSV challenge, these immune responses were further boosted and partial protection was obtained.
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Enhanced immunogenicity induced by an alphavirus replicon-based pseudotyped baculovirus vaccine against porcine reproductive and respiratory syndrome virus.
J. Virol. Methods
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Pseudotyped baculovirus has emerged as a promising vector for vaccine development and gene therapy. Alphaviruses, such as Semliki Forest virus (SFV), have also received considerable attention for use as expression vectors because of their self-replicating properties. In this study, pseudotyped baculovirus containing the hybrid cytomegalovirus (CMV) promoter/SFV replicon was used as a vector to co-express the GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV). The immunogenicity of the resulting recombinant baculovirus (BV-SFV-5m6) was compared with the pseudotyped baculovirus vaccine (BV-CMV-5m6), in which the expression of GP5 and M were driven by the CMV promoter only. In vitro, BV-SFV-5m6 exhibited enhanced expression of foreign proteins and also caused apoptosis in transduced cells. After immunization in BALB/c mice, BV-SFV-5m6 induced strong GP5-specific ELISA antibodies and neutralizing antibodies against homologous and heterologous viruses, along with dose sparing. Further analysis of the cell-mediated immune response showed that BV-SFV-5m6 elicited a Th1-dominant immune response that was greater than that elicited by BV-CMV-5m6. Taken together, the results of this study indicate that a baculovirus containing the hybrid CMV promoter/alphavirus replicon can be utilized as an alternative strategy to develop an efficacious vaccine against PRRSV infection.
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Porcine reproductive and respiratory syndrome virus infection activates IL-10 production through NF-?B and p38 MAPK pathways in porcine alveolar macrophages.
Dev. Comp. Immunol.
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Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging animal virus that has caused high economic losses for the swine industry worldwide. Previous in vitro and in vivo studies demonstrated that PRRSV infection induces significant production of interleukin 10 (IL-10), a pleiotropic cytokine with immuno-modulatory functions involved in host defense. However, the underlying regulatory mechanisms during PRRSV remain largely unknown. In the present study, we analyzed the expression kinetics of IL-10 in PRRSV-infected primary porcine alveolar macrophages (PAMs) and showed that PRRSV infection induced IL-10 mRNA and protein expression in a time- and dose-dependent manner. Inhibition of various molecules of the Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling pathways demonstrated that the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) has an important role in IL-10 induction during PRRSV infection. Furthermore, treatment with specific inhibitors or siRNA knockdown assays demonstrated that NF-?B and p38 MAPK (mitogen-activated protein kinase) are required for PRRSV-induced IL-10. Taken together, PRRSV infection significantly induced IL-10 expression and this induction depends on NF-?B activation and p38 MAPK in PAMs.
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Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102 isolated from a suckling piglet with acute diarrhea in China.
J. Virol.
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A diarrhea outbreak caused by porcine epidemic diarrhea virus (PEDV) has been observed in China since December 2010. We report here the complete genome sequence of PEDV strain AJ1102 isolated from a suckling piglet with acute diarrhea, which will help toward understanding the molecular and evolutionary characteristics of the epidemic PEDV in China.
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Complete genome sequence of a street rabies virus isolated from a rabid dog in China.
J. Virol.
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A rabies virus (RABV) was isolated from a dog in Anhui Province, China, in 2008. The virus was designated DRV-AH08. Its entire genome was sequenced and found to be closely related to RABV recently isolated in China and other Asian countries (homology of 87 to 98%) but distantly related to RABV in the "cosmopolitan" group (homology of 84 to 85%) in the clade I of RABV.
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A DNA vaccine encoding the FMDV capsid precursor polypeptide P1 and the enhancing effect of bovine herpesvirus 1 VP22 protein as molecular adjuvant.
Acta Virol.
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DNA vaccines containing the capsid precursor polypeptide P1 gene of foot-and-mouth disease virus (FMDV) alone or combined with the VP22 gene of bovine herpesvirus 1 (BVP22) as molecular adjuvant were constructed and used for immunization of BALB/c mice. The latter were challenged with FMDV and their humoral as well as cell-mediated immune responses and virus clearance capacity were assayed. Both DNA vaccines elicited specific immune responses, however, the DNA vaccine with the BVP22 adjuvant showed stronger responses and more efficient virus clearance. A stronger Th1 response was indicated by the IgG2a/IgG1 ratio. These results indicate that (i) a DNA vaccine based on FMDV P1 can stimulate significant immune responses and virus clearance and (ii) BVP22 is a potentially useful molecular adjuvant for such a vaccine. Keywords: DNA vaccine; foot-and-mouth disease virus; bovine herpesvirus 1.
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Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling.
J. Virol.
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Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-?/? response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-?B) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-?B and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.
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Complete genome sequence of porcine kobuvirus strain WUH1.
J. Virol.
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Porcine kobuvirus, an emerging virus, was first identified in Hungary in 2007. We report here the complete genome sequence of porcine kobuvirus strain WUH1 isolated from piglets with severe diarrhea, which will help toward understanding the molecular and evolutionary characteristics of the porcine kobuvirus.
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Pathogenesis of nonsuppurative encephalitis caused by highly pathogenic Porcine reproductive and respiratory syndrome virus.
J. Vet. Diagn. Invest.
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Since 2006, an unprecedented epidemic of highly pathogenic Porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection has emerged and prevailed in mainland China, causing so called high fever disease with a nervous symptom that is different from typical porcine reproductive and respiratory syndrome. To investigate this syndrome, the brains of pigs inoculated with HP-PRRSV were analyzed. The nucleic acid of HP-PRRSV was detected in brains by reverse transcription polymerase chain reaction. Histological examination demonstrated nonsuppurative encephalitis with lymphohistiocytic perivascular cuffing and infiltration of these leukocytes into the neuropil. Furthermore, transmission electron microscopy revealed that the HP-PRRSV that infected the endothelial cells crossed the blood-brain barrier into the central nervous system then induced cellular damage to neurons and neuroglial cells. These results provide a general insight into the pathway of HP-PRRSV invasion into brain tissue and the pathogenesis of nonsuppurative encephalitis.
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Porcine reproductive and respiratory syndrome virus nonstructural protein 2 contributes to NF-?B activation.
Virol. J.
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Nuclear factor-kappaB (NF-?B) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-?B in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) protein was identified as an NF-?B activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) contribute to NF-?B activation. In this study, we identified which Nsps can activate NF-?B and investigated the potential mechanism(s) by which they act.
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Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-genome.
PLoS ONE
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Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages.
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Complete genome sequence of porcine reproductive and respiratory syndrome virus isolated from piglet stool samples.
J. Virol.
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WUH4 is a highly pathogenic North American porcine reproductive and respiratory syndrome virus (PRRSV). Unlike previous PRRSV isolates, which were mainly recovered from sera or tissues, WUH4 was isolated from a piglet stool sample. Here we announce its complete genome sequence.
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Immunogenicity of foot-and-mouth disease virus structural polyprotein P1 expressed in transgenic rice.
J. Virol. Methods
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Transgenic plants have become developed as bioreactors for producing heterologous proteins and may even form edible vaccines. In the present study, a transgenic rice expressing the capsid precursor polypeptide (P1) gene of foot-and-mouth disease virus (FMDV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, northern blot, western blot, and ELISA analyses confirmed that the P1 gene was integrated into the transgenic rice and the protein was expressed specifically in the leaves at levels of 0.6-1.3 ?g/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, FMDV-specific neutralizing antibodies were detected. The immunized mice could clear virus from their sera after FMDV challenge. In addition, FMDV-specific mucosal immune responses were detected in mice after oral immunization with protein extracts from transgenic rice plants. Partial virus clearance was obtained after FMDV challenge. These results indicate the potential of using a transgenic rice-based expression system as an alternative bioreactor for FMDV subunit vaccines.
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Genome sequence of a highly prevalent porcine partetravirus in Mainland China.
J. Virol.
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Partetravirus is a novel defined genus of animal parvoviruses. Here, we first report the genome sequence of porcine partetravirus strain JSNJ62, which is highly prevalent in mainland China. It will help in understanding the epidemiology and molecular characteristics of the porcine partetravirus.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.