Emergence of Novel Reassortant H3N2 Avian Influenza Viruses in Southern China: Genetic Complexity and Pathogenicity in Chickens and Mice

H3N2 subtype avian influenza virus (AIV) is prevalent in poultry and wild birds and typically causes asymptomatic or mild respiratory infections. However, genetic reassortment between H3N2 and other AIV subtypes generates novel strains capable of crossing the species barrier, posing a threat to both poultry and public health. In this study, nine H3N2 AIVs were isolated from ducks in live poultry markets (LPMs) in Guangxi, southern China, during 2022–2024. Phylogenetic analysis revealed that all eight gene segments of the nine isolates were clustered within the Eurasian lineage, with internal genes derived from multiple subtypes, including H1, H2, H3, H4, H5, H6, H7, and H9. These findings indicate complex gene reassortment of H3N2 AIVs in Guangxi. Importantly, the PB2 genes of certain isolates were closely related to those of highly pathogenic H5 subtype viruses, suggesting that H3N2 AIVs may contribute internal genes to H5 viruses. Three representative isolates (LZD44, NND98, and NND100) were assessed for pathogenicity in SPF chickens and mice. All three strains successfully replicated in the respiratory tissues of both species. Notably, the LZD44 virus, which harbored the mammalian-adaptive mutations PB2-MVV and NP-I353V, presented significantly higher virulence in chickens and mice than the other two strains. These results demonstrate that H3N2 subtype AIVs are capable of replicating in certain tissues of chickens and mice without prior adaptation, underscoring a potential risk for cross-species transmission. Consequently, sustained surveillance of H3N2 subtype AIVs is essential to prevent the spillover of novel recombinants into the human population.