Introduction: In April 2025, three suspected human cases of avian influenza were identified in Changsha, China. Laboratory testing confirmed three cases of H9N2 AIV infection. This report summarizes the epidemiological findings from cases and contact investigations, along with genetic characterization of the isolated H9N2 strains.

Methods: Comprehensive epidemiological assessments were conducted for each confirmed case. Virus isolation and culture were performed using throat swab specimens obtained from the cases. Isolated H9N2 strains were sequenced using next-generation sequencing (NGS). HA and NA gene sequences were analyzed for homology; evolutionary trees were constructed; and key antigenic sites were examined to identify genetic features.

Results: All three cases were sporadic. No influenza-like illness was observed among close contacts or live poultry store employees during the 10-day medical monitoring period. Phylogenetic analysis indicated that the HA gene of all three H9N2 strains belonged to the A/Duck/Hong Kong/Y280/97 (Y280-like) clade within the Eurasian lineage. HA gene sequence homology was 99.7%–99.8%, and NA gene homology was 98.4%–99.8%. The HA protein cleavage site was identified as PSRSSR↓GLF. Several HA protein site mutations were detected — H191N, A198T/V, Q226L, and Q234L — that had been previously associated with increased binding to receptors. HA-232H, 234L, and 236G support a binding preference for the human-type sialic acid-α-2,6-galactose receptors.

Conclusion: All three H9N2 avian influenza cases were mild and involved reported exposure to poultry or related environments. Genetic analysis revealed high homology of HA and NA among the isolated viruses. No epidemiological links were identified between cases, and no evidence was found of sustained human-to-human transmission. Continued avian influenza surveillance and public health education are warranted.