Objective To analyze the genetic evolution and molecular characteristics of avian influenza A (H9N2) virus in the external environment of live poultry markets in Changsha, Hunan province, from 2021 to 2024, understand the epidemiological characteristics and transmission mechanisms of H9N2 virus in these markets and provide evidence for the development of avian influenza prevention and control measures.

Methods Avian influenza viruses in samples were detected by using quantitative real-time PCR, and H9N2 virus strains were isolated through chicken embryo culture. The whole genomes of the isolates were sequenced by using next-generation sequencing platform, and the sequencing results were aligned and analyzed for genetic evolution with software MEGA 7 .

Results From 2021 to 2024, a total of 441 environmental samples were collected from the live poultry markets in Changsha, of which 373 (84.58%) were positive for influenza A virus and 356 (80.73%) were positive for subtype H9. After the positive specimens were inoculated with chicken embryos, 17 H9N2 virus strains were successfully isolated. Genetic evolution analysis showed that the hemagglutinin (HA) and neuraminidase (NA) genes of the 17 isolates all belonged to the Y280-like lineage of the Eurasian type of avian influenza A (H9N2) virus and all belonged to the G57 genotype. Amino acid sequence analysis showed that there was no continuous insertion of basic amino acids in the HA protein cleavage site, which was consistent with the typical characteristics of low pathogenic avian influenza virus. Mutations associated with enhanced cross-species infection and mammalian pathogenicity were found at multiple key amino acid loci, including H183N, A190T/V, and Q226L in HA protein, amino acid deletions at positions 63−65 in the stem of NA protein, A588V and E627V in polymerase basic protein 2, I368V and D622G in polymerase basic protein 1, K356R and S409N in polymerase acid protein, I353V in nuclear protein, N30D in matrix protein 1, and P42S in nonstructural protein 1. In addition, the S31N mutation was detected in the matrix protein 2 of 16 isolates, suggesting that these strains are resistant to adamantane.

Conclusion The positive rate of avian influenza virus was high in the environment of live poultry markets in Changsha, and the isolated strains pose a significant risk of cross-species transmission. It is necessary to strengthen the surveillance in live poultry markets and pay closely attention to the molecular evolution of the virus.