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ABSTRACTMacroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. The protein degradation role of autophagy has been widely used to control viral infection at multiple levels. In the ongoing evolutionary arms race, viruses have developed various ways to hijack and subvert autophagy in favor of its replication. It is still unclear exactly how autophagy affects or inhibits viruses. In this study, we have found a novel host restriction factor, HNRNPA1, that could inhibit PEDV replication by degrading viral nucleocapsid (N) protein. The restriction factor activates the HNRNPA1-MARCHF8/MARCH8-CALCOCO2/NDP52-autophagosome pathway with the help of transcription factor EGR1 targeting the HNRNPA1promoter. HNRNPA1 could also promote the expression of IFN to facilitate the host antiviral defense response for antagonizing PEDV infection through RIGI protein interaction. During viral replication, we found that PEDV can, in contrast, degrade the host antiviral proteins HNRNPA1 and others (FUBP3, HNRNPK, PTBP1, and TARDBP) through its N protein through the autophagy pathway. These results reveal the dual function of selective autophagy in PEDV N and host proteins, which could promote the ubiquitination of viral particles and host antiviral proteins and degradation both of the proteins to regulate the relationship between virus infection and host innate immunity.Abbreviations:3-MA: 3-methyladenine; ATG: autophagy related; Baf A1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4′,6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PEDV: porcine epidemic diarrhea virus; siRNA: small interfering RNA; TCID50: 50% tissue culture infectious doses.

ABSTRACTSenecavirus A (SVA), an important emerging porcine virus, has outbreaks in different regions and countries each year, becoming a virus with global prevalence. SVA infection has been reported to induce macroautophagy/autophagy; however, the molecular mechanisms of autophagy induction and the effect of SVA on autophagy remain unknown. This study showed that SVA infection induced the autophagy process in the early stage of SVA infection, and the rapamycin-induced autophagy inhibited SVA replication by degrading virus 3 C protein. To counteract this, SVA utilized 2AB protein inhibiting the autophagy process from promoting viral replication in the late stage of SVA infection. Further study showed that SVA 2AB protein interacted with MARCHF8/MARCH8 and LC3 to degrade the latter and inhibit the autophagy process. In addition, we found that MARCHF8 was a positive regulator of type I IFN (IFN-I) signaling. During the autophagy process, the SVA 2AB protein targeted MARCHF8 and MAVS forming a large complex for degradation to deactivate IFN-I signaling. Together, our study reveals the molecular mechanisms of selective autophagy in the host against viruses and reveals potential viral strategies to evade the autophagic process and IFN-I signaling for successful pathogenesis.Abbreviations:Baf A1: bafilomycin A1; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4′,6-diamidino-2-phenylindole; hpi: hours post-infection; IFN: interferon; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane associated ring-CH-type finger 8; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; Rapa: rapamycin; RT: room temperature; siRNA: small interfering RNA; SVA: Senecavirus A; TCID50: 50% tissue culture infectious doses.

ABSTRACTInterferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway.Abbreviations: 3MA: 3-methyladenine; ATG: autophagy-related; Baf A1: bafilomycin A1; BST2: bone marrow stromal cell antigen 2; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; CT: cytoplasmic tail; DAPI: 4′,6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; IRF1: interferon regulatory factor 1; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PED: porcine epidemic diarrhea; PEDV: porcine epidemic diarrhea virus; RT: room temperature; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription; TCID50: 50% tissue culture infectious doses; TM: transmembrane.

ABSTRACTMonoclonal antibodies (MAbs) against Haemophilus parasuiswere generated by fusing spleen cells from BALB/c mice immunized with whole bacterial cells with SP2/0 murine myeloma cells. Desirable hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Neutralizing MAb 1D8 was selected in protection assays. ELISA results demonstrated that 1D8 can react with all 15 serotypes of H. parasuisand field isolate H. parasuisHLJ-018. Passive immunization studies showed that mice inoculated intraperitoneally with 1D8 had significantly reduced prevalence of H. parasuiscolonization in the blood, lung, spleen, and liver and had prolonged survival time compared to that of the control group. Furthermore, the passive transfer experiment indicated that MAb 1D8 can protect mice from both homologous and heterologous challenges with H. parasuis. Using two-dimensional gel electrophoresis (2-DE), the immunoreactive protein target for MAb 1D8 was identified. The data presented confirm the protective role of MAb 1D8 and identify OmpA as the target of the protective monoclonal antibody. The data suggest that OmpA is a promising candidate for a subunit vaccine against H. parasuis.

A rapid immune colloidal gold test strip for detecting 1 lineage of the H5 subtype of highly pathogenic avian influenza was developed based on membrane chromatography with monoclonal antibodies (mAbs) against hemagglutinin (HA) of the H5 subtype of Avian influenza virus (AIV). Bagg albino C mice that were immunized with AIV A/Goose/Guangdong/3/96(H5N1) yielded 4 hybridomas secreting mAbs (1A6, 1B8, 3C4, and 8F6) specific for HA. Monoclonal antibody 3C4 was conjugated with colloidal gold as the detector antibody. On the test strip, 1A6, in combination with rabbit anti-H5N1 polyclonal antibody, was used as the capture complex at the “test line,” and goat antimouse immunoglobulin G antibody was used as the capture antibody at the “control line.” The strip test correctly detected the H5 subtype of AIV in known positive samples and did not react with the standard antigens of AIV H1-H4 or H6-H14, or the antigens of Newcastle disease virus, Infectious bursal disease virus, Infectious bronchitis virus,or Avian infectious laryngotracheitis virus. When 483 clinical specimens suspected of infection with H5N1 were tested, results from the strip test were nearly identical with those from the HA/hemagglutination inhibition test, reverse transcription polymerase chain reaction, and a commercial enzyme-linked immunosorbent assay kit. Because the newly developed strip was specific, rapid, and sensitive, it should be useful for diagnosing the H5 subtype of AIV and for investigating its epidemiology. The test strip is only reliable for the lineage of H5N1. As for the other strains of AIV, further experiments will be needed.

The σC-encoding gene of two muscovy duck reovirus (DRV) S14 and C4 strains were cloned and completely sequenced. The open reading frame (ORF) comprised 810 bp and encoded 269 amino acids with a predicated molecular mass of 29.5 kDa. Expressed σC fusion protein in Escherichia coliBL21 strain could be detected by Western blotting under duck anti-reovirus polyclonal serum. There are two large gap insertions at the N-terminal part of the DRV σC when necessary to optimize the alignment of the amino acid sequences of the DRV σC had a heptapeptide repeat and leucine zipper patterns structurally related to ARV σC. All DRVs grouped into one specified genogroup within Orthoreovirusesgenus subgroup II. The degree of differences between the S14/C4 and ARV was only 23–24%, and 21–22%, respectively, at both the nucleotide and deduced amino acid levels, suggested that DRVs are quite different from ARVs and should give a precise classification for DRVs in Orthoreovirusgenus.The σC-encoding gene of two muscovy duck reovirus (DRV) S14 and C4 strains were cloned and completely sequenced. The open reading frame (ORF) comprised 810 bp and encoded 269 amino acids with a predicated molecular mass of 29.5 kDa. Expressed σC fusion protein in Escherichia coliBL21 strain could be detected by Western blotting under duck anti-reovirus polyclonal serum. There are two large gap insertions at the N-terminal part of the DRV σC when necessary to optimize the alignment of the amino acid sequences of the DRV σC had a heptapeptide repeat and leucine zipper patterns structurally related to ARV σC. All DRVs grouped into one specified genogroup within Orthoreovirusesgenus subgroup II. The degree of differences between the S14/C4 and ARV was only 23–24%, and 21–22%, respectively, at both the nucleotide and deduced amino acid levels, suggested that DRVs are quite different from ARVs and should give a precise classification for DRVs in Orthoreovirusgenus.

High-level expression of the ORF6gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6gene of PRRSV and found that it could not be expressed in Pichia pastorisstrain GS115. Then, the ORF6gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5′-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZαA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6gene.