Department of Veterinary Medicine
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Item A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus(MDPI, 2016-08-18) Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, YanjinHepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology.Item The Capsid Protein of Hepatitis E Virus Inhibits Interferon Induction via Its N-Terminal Arginine-Rich Motif(MDPI, 2019-11-11) Lin, Shaoli; Yang, Yonglin; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yan-JinHepatitis E virus (HEV) causes predominantly acute and self-limiting hepatitis. However, in HEV-infected pregnant women, the case fatality rate because of fulminant hepatitis can be up to 30%. HEV infection is zoonotic for some genotypes. The HEV genome contains three open reading frames: ORF1 encodes the non-structural polyprotein involved in viral RNA replication; ORF2 encodes the capsid protein; ORF3 encodes a small multifunctional protein. Interferons (IFNs) play a significant role in the early stage of the host antiviral response. In this study, we discovered that the capsid protein antagonizes IFN induction. Mechanistically, the capsid protein blocked the phosphorylation of IFN regulatory factor 3 (IRF3) via interaction with the multiprotein complex consisting of mitochondrial antiviral-signaling protein (MAVS), TANK-binding kinase 1 (TBK1), and IRF3. The N-terminal domain of the capsid protein was found to be responsible for the inhibition of IRF3 activation. Further study showed that the arginine-rich-motif in the N-terminal domain is indispensable for the inhibition as mutations of any of the arginine residues abolished the blockage of IRF3 phosphorylation. These results provide further insight into HEV interference with the host innate immunity.Item A NOVEL INTERFERON-INDUCING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS STRAIN: CHARACTERIZATION AND VACCINE DEVELOPMENT(2018) Ma, Zexu; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Porcine reproductive and respiratory syndrome virus (PRRSV) causes a swine infectious disease characterized by severe reproductive failure in sows and respiratory disease in pigs of all ages. Despite substantial efforts to control PRRS, no production or vaccination regimen has demonstrated sustaining success. Type I interferons (IFNs) are critical to the innate immunity against viral infections and play an important role in activation of the adaptive immune response. PRRSV appears to antagonize induction of type I IFNs. Fortunately, we discovered an atypical PRRSV strain, A2MC2, which induces type I IFNs in cultured cells. A2MC2 elicits earlier onset and higher levels of virus-neutralizing antibodies than the Ingelvac PRRS® MLV in pigs. However, moderate virulence of A2MC2 was observed in infected piglets. The objective of this project was to characterize A2MC2 and explore this unique strain for the development of an improved vaccine against PRRS. First, I attenuated this strain by serial passaging in MARC-145 cells for 90 consecutive passages. The passage 90 virus (A2MC2-P90) was avirulent and retained the capability of IFN induction. The A2MC2-P90 virus induced higher level virus-neutralizing antibodies in pigs. Secondly, I constructed an infectious cDNA clone of A2MC2. The recovered virus from the infectious clone was similar to the parental strain in growth properties and IFN induction. Gene fragment swapping demonstrated that the middle half genome of A2MC2 was essential for its IFN induction. Thirdly, I conducted studies to exam the genetic source of A2MC2 in IFN induction. Comparison of A2MC2 and other closely relevant PRRSV strain identifies five unique non-synonymous nucleotides. These five nucleotides remained unchanged in the A2MC2-P90 virus. Site-directed mutagenesis indicated that one unique nucleotide in A2MC2 genome was critical in the IFN induction as mutation of this nucleotide led to the loss of IFN induction. Together, our data demonstrate that A2MC2 is a novel strain that is worth further exploration for an improved vaccine against PRRS. The infectious clone of A2MC2 will be useful for the development of a marker vaccine by insertion of a marker sequence into the A2MC2 genome.Item Inducing Autophagic Cell Death by Nsp5 of Porcine Reproductive and Respiratory Syndrome Virus(Austin Publishing Group, 2015-11-10) Yang, Liping; Wang, Rong; Ma, Zexu; Wang, Yu; Zhang, YanjinPorcine Reproductive and Respiratory Syndrome (PRRS) leads to severe economic losses to the swine-producing industry. Many unclear questions remain on pathogenesis of PRRS virus (PRRSV), including the mechanism of PRRSV-induced cell death. In this study, we cloned and expressed a PRRSV non-structural protein, nsp5, and discovered that it induced cell death in cultured cells. The nsp5 protein localized in cytoplasm and majority of the protein concentrated in perinuclear region. Along with extension of incubation time, the nsp5 tended to form puncta and polarized besides nucleus. An interesting observation was that the nsp5 expression induced cell death. Cell viability assay showed that the cells with nsp5 expression had over 2-fold more cell death than cells with empty vector. Further study indicated that the nsp5 induced cell death via autophagy. Treatment with 3-MA, an autophagy inhibitor, blocked the nsp5- induced cell death. These results suggest that nsp5 might play an important role in PRRSV-induced cell death. Further examination on the mechanism is warranted.Item Sustaining Interferon Induction by a High-Passage Atypical Porcine Reproductive and Respiratory Syndrome Virus Strain(Nature Publishing Group, 2016-11-02) Ma, Zexu; Yu, Ying; Xiao, Yueqiang; Opriessnig, Tanja; Wang, Rong; Yang, Liping; Nan, Yuchen; Samal, Siba K.; Halbur, Patrick G.; Zhang, Yan-JinPorcine reproductive and respiratory syndrome virus (PRRSV) strain A2MC2 induces type I interferons in cultured cells. The objective of this study was to attenuate this strain by serial passaging in MARC-145 cells and assess its virulence and immunogenicity in pigs. The A2MC2 serially passaged 90 times (A2MC2-P90) retains the feature of interferon induction. The A2MC2-P90 replicates faster with a higher virus yield than wild type A2MC2 virus. Infection of primary pulmonary alveolar macrophages (PAMs) also induces interferons. Sequence analysis showed that the A2MC2-P90 has genomic nucleic acid identity of 99.8% to the wild type but has a deletion of 543 nucleotides in nsp2. The deletion occurred in passage 60. The A2MC2-P90 genome has a total of 35 nucleotide variations from the wild type, leading to 26 amino acid differences. Inoculation of three-week-old piglets showed that A2MC2-P90 is avirulent and elicits immune response. Compared with Ingelvac PRRS® MLV strain, A2MC2-P90 elicits higher virus neutralizing antibodies. The attenuated IFN inducing A2MC2-P90 should be useful for development of an improved PRRSV vaccine.