H5 low pathogenic avian influenza viruses maintained in wild birds in China.

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.