Abstract
Background: H9N2 avian influenza viruses (AIVs) donate their genes to other subtype AIVs, posing significant threats to poultry industries and public health due to their endemicity and zoonotic potential. This study investigates the molecular evolution, reassortment, and mutations of the H9N2 isolates from the live poultry markets (LPMs) in Xinjiang, China. Methods: AIVs were isolated from oropharyngeal and cloacal swabs, as well as environmental samples collected during the winter of 2017-2018. Full-genome sequencing and phylogenetic and molecular analysis were conducted to elucidate viral origins, reassortment patterns, and molecular characteristics. Results: Thirty H9N2 isolates were obtained, all belonging to the G57 genotype. Phylogenetic analysis revealed three distinct Eurasian lineages: BJ/94-like (HA, NA), G1-like (PB2, MP), and F98-like. Notably, viral genes diverged into two major branches (A/B), with the A branches of HA, PB2, PA, and NS further subdivided into A1/A2 sublineages. In addition to the A and B branches, the viral genes of several isolates formed independent phylogenetic branches. Some of the viral genes clustered together with H9N2 viruses from the poultry/environmental strains in China and Japan, and some viral genes (e.g., PB2, PB1, MP, and NS) showed close phylogenetic relationships with human-infecting H9N2/H7N9 viruses. The multiple mutations detected in the isolates were associated with viral virulence, mammalian adaptation, and transmission. Conclusion: Xinjiang H9N2 viruses display complex reassortment dynamics involving multiple geographic lineages. Their genetic connection to human-infecting strains underscores the risk of zoonotic spillover. Enhanced surveillance in LPMs is crucial for pandemic preparedness.