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• Three subtype H5 avian influenza virus strains (H5N2, H5N3 and H5N8), were isolated from wild birds in China in 2020. • The viruses have undergone complicate reassortment by acquiring genes from AIVs of both Eurasian and North American lineages. • These H5 viruses derived from wild birds can replicate and transmit efficiently in ducks. • These H5 viruses have the ability to replicate in mammalian cells, and can replicate in BALB/c mice without prior adaptation. Low pathogenic avian influenza virus, H5 or H7 subtype, possesses the potential capability to change to highly pathogenic variant, which damages wild waterfowl, domestic poultry, and mammalian hosts. In regular active surveillance of avian influenza virus from wild birds in China in 2020, we isolated six H5 avian influenza viruses, including one H5N2, two H5N3, and three H5N8. Phylogenetic analysis indicated that the H5N2 and H5N3 isolates clustered into Eurasian lineage, whereas the H5N8 viruses were originated in North America. The HA proteins of six viruses carried the cleavage-site motif PQRETR↓GLF, which indicated low pathogenicity of the viruses in chickens. However, the N30D, I43M, and T215A mutations in M1 protein and the P42S, I106M, and C138F residues changed in NS1 protein, implying all viruses could exhibit increased virulence in mice. Viral replication kinetics in mammalian cells demonstrated that the three representative viruses had the ability to replicate in both MDCK cells and A549 cells with low titers. Even though two of three representatives, WS/SX/S3-620/2020(H5N3) and ML/AH/A3-770/2020(H5N8), did not replicate and transmit efficiently in poultry (chickens), they did replicate and transmit efficiently in waterfowl (ducks). Viral pathogenicity in mice indicated that both H5N2 and H5N3 viruses are able to replicate in the nasal turbinates and lungs of mice without prior adaptation, while the H5N8 virus could not. The intercontinental and cross-species transmission of viruses may continuously exist in China, thereby providing constant opportunities for virus reassortment with local resident AIVs. Thus, it is crucial to continuously monitor migration routes for AIVs by systematic surveillance. [ABSTRACT FROM AUTHOR]

• Three H6 subtype avian influenza virus strains (H6N1, H6N2 and H6N8), were isolated from wild birds in China from 2017 and 2019. • The viruses have undergone reassortment by acquiring genes from AIVs of both Eurasian and North American lineages. • These H6 viruses derived from wild birds can replicate efficiently in BALB/c mice without pre-adaptation. During our routine surveillance, we isolated seven H6 avian influenza virus (AIV) strains, including three H6N1 strains, three H6N2 strains, and one H6N8 strain, from 3667 fresh fecal samples that were collected from wild bird habitats in China from March 2017 and May 2019. Phylogenetic analysis revealed that these viruses formed five different genotypes and have undergone complicate reassortment during their evolution by acquiring genes from AIVs of both Eurasian and North American lineages that have been previously detected in migrating waterfowl and poultry. Viral pathogenesis in mice showed that these H6 viruses replicated efficiently in both the nasal turbinates and lungs of mice without pre-adaptation, but none of them were lethal for mice. We studied the genetic characteristic and biological property of novel reassortant H6 viruses isolated from wild birds in China. It also highlights the need for continued surveillance of H6 AIVs circulating in nature. [ABSTRACT FROM AUTHOR]

• MDRV-induced autophagy can promote virus replication in DF-1 cells, and MDRV utilized the autophagosomes for replication. • MDRV infection can promote the fusion of the autophagosomes and lysosomes. • MDRV inhibits the maturation of lysosomes. • MDRV promotes self-replication by inducing and regulating autophagy processes. Autophagy plays a momentous role in cellular responses against pathogens. However, the influence of the autophagy machinery on Muscovy duck reovirus (MDRV) infection is not yet confirmed. In this study, it was shown that MDRV infection significantly increased the number of autophagy-like vesicles in DF-1 cells under electron microscope and the LC3-I/LC3-II conversion, which was considered important indicators of autophagy. It was worth noting that the level of autophagy was positively correlated with MDRV replication. Further test results showed that MDRV-induced autophagy can promote virus replication in DF-1 cells, and both the envelope protein sigma A and non-structural protein sigma NS that play an important role in virus replication process can colocalize with the autophagosome marker molecule LC3-II by confocal immunofluorescence analysis. These results indicated that MDRV utilized the autophagosomes for replication. Through transfection of the dual fluorescent plasmid mcherry-EGFP-LC3 and fluorescence microscope observation, it was found that autophagosomes were more likely to fuse with lysosomes in MDRV-infected cells compared with the blank group. The phenomenon of pEGFP-LC3B fluorescent spot and LAMP1 co-localization appeared in MDRV infected cells, indicating that MDRV infection would promote the fusion of autophagosomes and the lysosomes. Conversely, accumulation of p62 was observed by immunoblotting, suggesting that autolysosomes does not exert effective degradation. MDRV infection triggered a incomplete autophagic response. Further studies found that the expression of LAMP1, a marker protein of late endosome/early lysosome, increased significantly in MDRV-infected cells, suggesting an increase in the number of immature lysosomes. In addition, the experiment detected the maturation of the lysosomal acid hydrolase Cathepsin D in the cells, and found that the expression of the 33 kDa mature form of Cathepsin D was significantly reduced after MDRV infection, indicating that MDRV inhibits the maturation of lysosomes. In general, MDRV infection induces autophagy of DF-1 cells, promotes the fusion of autophagosomes and lysosomes, inhibits autophagolysosome degradation, and promotes virus replication. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a highly infectious and lethal viral disease caused by the African swine fever virus (ASFV). The four prominent loop structures on the surface of the primary structural protein P72 are considered to be key protective epitopes. In this study, the four critical loops (ER1–4) of the ASFV p72 protein were individually fused to hepatitis B virus core particles (HBc) and self-assembled into nanoparticles to preserve the natural conformation of the loop structure and enhance its immunogenicity. Then, four recombinant proteins were obtained in E. coli expression system and monoclonal antibodies (mAbs) were developed and characterized. All 10 mAbs obtained were able to react with P72 protein and ASFV with potencies up to 1:204 800. Amino acids 250–274, 279–299 and 507–517 of the P72 protein were identified as linear epitopes and highly conserved. The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. Importantly, neutralization experiments illustrated that mAb 4G8 has a 67% inhibition rate, indicating that its corresponding epitopes are potential candidates for ASFV vaccine. In conclusion, highly immunogenic nanoparticles of the ASFV P72 key loop were constructed to induce the production of highly effective mAbs and clarify their epitope information for the diagnosis and prevention of ASFV. • Four recombinant VLPs with preserved natural structure and enhanced immunogenicity were obtained. • High titers of monoclonal antibodies were induced with VLPs, and monoclonal antibody 4G8 had the highest antibody titer of 1:2048000. • Amino acids 250–274, 279–299 and 507–517 of the P72 protein were identified as linear epitopes and highly conserved. • The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. More importantly, the mAb 4G8 inhibited the replication of the virus by 67%, indicating that its corresponding epitopes are potential vaccine candidate antigens. [ABSTRACT FROM AUTHOR]

• The histopathological effects of Muscovy duck reovirus was examined in vivo. • The virus degrades the morphology and function of the small intestinal mucosa. • The virus alters the local density of immune-related cells. • The virus also alters the secretion of immune-related factors. Muscovy duck reovirus (MDRV) causes serious immunodeficiency in the intestinal mucosa, although the underlying histopathological mechanisms remain unclear. Thus, we investigated the impact of MDRV infection on intestinal morphology using hematoxylin and eosin staining. Immune-related cells were also quantified by staining with hematoxylin and eosin, toluidine blue, and periodic acid-Schiff stain, or by immunohistochemistry and cytochemistry for lectin. Similarly, CD4+ and CD8+ cells were quantified by flow cytometry, and the expression of several immune-related molecules was quantified by radioimmunoassay. We found that MDRV clearly damaged the intestinal mucosa, based on tissue morphology, villus length, villus width, intestinal thickness, villus height/crypt depth ratio, and villus surface area. MDRV also altered the density or distribution of lymphocytes, mastocytes, and goblet cells in the small intestinal mucosa, as well as microfold cells in Peyer's patches. In addition, MDRV markedly depleted CD4+ cells from the intestinal mucosa and lowered the CD4+:CD8+ ratio in peripheral blood. Moreover, MDRV diminished the levels of secretory IgA and mucosal addressin cell adhesion molecule-1 (p < 0.01), but elevated those of histamine and nitric oxide (p < 0.01 or p < 0.05). Finally, MDRV significantly suppressed IL-1β, IL-4, IL-5, and IL-8 levels (p < 0.01 or p < 0.05) mid-infection. Collectively, our data suggest that MDRV severely damages the structure and function of the intestinal mucosa by modulating immune cells and immune-related factors, thus leading to local immunodeficiency. Our findings lay the foundation for further research on the pathogenesis of MDRV. [ABSTRACT FROM AUTHOR]