Marked Antigenic Divergence and Evolutionary Analysis of H5 AIVs from Wild Birds in East China, 2013-2022

The highly pathogenic H5 subtype avian influenza viruses (AIVs) pose persistent threats to the poultry industry and public health owing to their high lethality and pandemic potential. Migratory wild birds play a pivotal role in the global dissemination and genetic reassortment of the virus, serving as both natural reservoirs and long-distance vectors that drive its spatiotemporal spread. However, the extent and evolutionary drivers of antigenic divergence among H5 AIVs circulating in wild birds in East China remain poorly understood. Here, we aim to characterize the evolutionary dynamics and antigenic divergence of H5 AIVs isolated from wild birds in East China between 2013 and 2022. Whole-genome sequencing and phylogenetic analysis revealed that the isolates belonged to multiple clades, including 2.3.2.1 and 2.3.4.4, and encompassed the H5N1, H5N6, and H5N8 subtypes. Key amino acid site analysis showed that the glycosylation site patterns in the HA and NA proteins varied among clades, with some strains exhibiting gains or losses of glycosylation sites, while certain strains had acquired mutations associated with mammalian adaptation. Cross-hemagglutination inhibition (HI) assays combined with antigenic cartography demonstrated that the majority of the isolates were antigenically well-matched with the contemporaneous vaccine strains used in China, indicating that these vaccines effectively covered the predominant circulating antigenic variants at the time. Nevertheless, potential antigenic mismatches were still observed between some circulating strains and these vaccine strains. These findings suggest that wild birds in East China may contribute to the regional movement and diversification of H5 AIVs, highlighting the value of sustained surveillance for early warning and vaccine strain evaluation.