Introduction: The H3N8 avian influenza virus (AIV) is recognized for its capacity for interspecies transmission and has been detected in multiple mammalian hosts. Between 2022 and 2023, three human infections with H3N8 were documented in China, raising significant concerns about its zoonotic spillover potential. In this study, we characterized an H3N8 isolate from Niukouyu Wetland Park in Beijing Municipality to elucidate the genetic variability and evolutionary dynamics of this AIV subtype.

Methods: The virus underwent whole-genome sequencing followed by comprehensive molecular and phylogenetic characterization.

Results: We identified a genetically reassorted, low-pathogenicity H3N8 AIV, marking the first detection of this subtype in a wild environment in Beijing. Throat swabs from the park staff tested negative for influenza viruses. Phylogenetic analyses demonstrated that the viral hemagglutinin and neuraminidase genes originated from Eurasian and North American lineages, respectively. Nucleotide sequence comparisons revealed 97.57%–99.06% similarity between the eight gene segments of this virus and those of reference strains. Multiple internal gene mutations were identified, including PB2-K318R and PB1-F2-N66S, which are associated with enhanced polymerase activity, increased virulence, and improved mammalian adaptation.

Conclusions: The molecular characteristics of this H3N8 virus indicate a potential risk for cross-species transmission to humans, emphasizing the critical need to strengthen influenza surveillance networks and expand monitoring efforts targeting H3 subtype AIVs.