Objective To isolate viruses from human H9N2 infection cases and concurrent environmental positive samples in Shiyan City in 2024, conduct whole-genome sequencing and analysis, evaluate the cross-species transmission risk of the virus, and provide evidence for local prevention and control.
Methods Whole-genome next-generation sequencing was carried out on one human-derived strain and four environmental strains. Phylogenetic analysis, homology analysis, key amino acid mutation analysis of the HA/NA genes, as well as temporal divergence and population dynamics analysis of the HA gene, were conducted.
Results All five strains belonged to the H9N2 Beijing/1/94 lineage. The nucleotide and amino acid homologies of the HA gene were 98.25%-99.87% and 99.03%-100%, respectively; for the NA gene of the three strains, the nucleotide and amino acid homologies were 99.57%-99.93% and 99.57%-100%, respectively. Analysis showed that the time to the most recent common ancestor (TMRCA) of the HA gene of 201 H9N2 strains circulating in China from 2020 to 2025 was 1982.74, with an evolutionary rate of 4.11×10?3 substitutions/site/year (95% confidence interval: 3.695×10?3–4.537×10?3); The divergence time of the HA gene of the current strains was 2021.61. Population dynamics analysis revealed a stable population size from 1982 to 2015, followed by a gradual increase after 2015, an explosive growth in 2022, a peak in 2023, and a rapid decline thereafter. The HA cleavage site motif was PSRSSR↓GLF in all strains (low-pathogenicity feature). Mutations in the receptor-binding domain included S158N/D, S/F/A160N, H183N, E190V, Q226L, and Q227M. Compared with classic reference strains, the 105NGT and 218NRT sites were deleted, while a new 313NCS site was added, resulting in a total of seven conserved potential N-glycosylation sites. Three strains showed a deletion of TEI at positions 63–65 in the NA protein stalk, and mutations K368N and D369S in the erythrocyte-binding site; no mutations were found at neuraminidase inhibitor (oseltamivir and zanamivir) resistance sites.
Conclusion This human infection originated from exposure in live poultry markets. The circulating strains have acquired key mutations that enhance human-type receptor binding ability, showing significant genetic evolution. It is recommended to strengthen routine surveillance and disinfection of live poultry markets, continuously conduct whole-genome monitoring of human and environmental strains, closely track mutations and drug resistance dynamics, and provide timely early warnings.