Abstract
The H4 subtype of avian influenza virus (AIV) is prevalent worldwide, but only receives little attention due to its low pathogenicity in poultry. Consequently, it remains largely unclear whether H4 AIVs pose a potential threat to the poultry industry and public health. During the period from 2011 to 2022, we conducted an active surveillance programm. A total of 154,762 swab samples were collected across various provinces, and 427 H4 viruses were detected, resulting in a positivity rate of 0.28%. All H4 viruses were isolated from poultry, primarily from ducks in live poultry markets. We further investigated the genetic evolutionary characteristics and pathogenicity of 20 H4Nx viruses isolated in our program. Phylogenetic analysis revealed that the 20 H4Nx viruses belonged to the Eurasian lineage and exhibited significant genetic diversity, with 19 distinct genotypes identified. Molecular characterization indicated that these viruses were low-pathogenicity AIVs with limited binding affinity to human receptors, yet they contained mutations associated with enhanced viral replication and pathogenicity in mammals. Pathogenicity tests conducted in ducks demonstrated that H4 viruses were weakly pathogenic, exhibiting limited replication and transmission capabilities. However, some viruses were able to replicate effectively in mice and induce weight loss. For instance, DK/AH/AG61/11(H4N6) can replicate efficiently in MDCK cells, indicating a potential threat to mammals. These findings underscore the importance of ongoing surveillance of H4 AIVs to better understand their evolution and transmission dynamics and to prevent potential public health risks.