Genetic characterization and evolution of H6N6 subtype avian influenza viruses.

H6-subtype avian influenza virus (AIV) was prevalent in the world and could sporadically infect humans. Here, a new chicken-derived H6N6-subtype AIV strain A/chicken/Zhejiang/49/2021 (ZJ49) was isolated in Zhejiang Province, China in 2021. Phylogenetic analysis by Maximum likelihood methods showed that H6-subtype AIVs were classed into 13 groups according to HA gene. The ZJ49 strain belonged to the G12 group, which mainly consisted of strains from Asian and dominated in recent years. Based on NA gene, H6-subtype AIVs were divided into N6.1 and N6.2 clades according to the NA gene. The ZJ49 isolate was located in the N6.2e clade, which mainly consisted of the H5N6-subtype AIVs. Phylogenetic analysis by Bayesian methods showed that the effective quantity size of H6-subtype AIVs increased around 1990, reached a peak around 2015, declined after 2015, then kept in a stable level after 2018. The reassortment analysis predicted that the PB2, PA, and NA genes of ZJ49 may recombine with H5-subtype AIVs. The amino acid at 222 position of HA gene of ZJ49 strain mutated from A to V, suggesting that ZJ49 has a potential ability to cross species barriers. The four glycosylation sites were highly conserved, implying less impact on the fold and conception of HA stem structure. Our results revealed the complicated evolution, reassortment, and mutations of receptor binding sites of H6-subtype AIVs, which emphasize the importance to continuously monitor the epidemiology and evolution of H6-subtype AIVs.