Molecular epidemiological characteristics of H9N2 subtype avian influenza virus in the external environment of western Zhejiang, China, 2014–2025
TL;DR
The external environment in western Zhejiang exhibits a high prevalence of AIVs with pronounced spatiotemporal clustering, and the H9N2 subtype AIV has accumulated multiple mammalian adaptive mutations, and exhibits genetic linkages across eastern Chinese provinces.
Abstract
Objective To elucidate the epidemiological distribution patterns of avian influenza virus (AIV) in the external environment of western Zhejiang from 2014 to 2025, analyze the molecular epidemiological characteristics of the H9N2 subtype, and assess its public health risks. Methods According to the Zhejiang Provincial Surveillance Program for Avian Influenza in Occupationally Exposed Populations and External Environments, real-time RT-PCR was used to detect AIV subtypes in environmental specimens. H9N2-positive samples with cycle threshold values <30 were inoculated into specific pathogen-free (SPF) embryonated chicken eggs for virus isolation, followed by whole-genome sequencing and bioinformatics analysis for phylogenetic and molecular characterization. Results A total of 7,762 specimens were tested from 2014 to 2025, with an overall positivity rate of 34.64% (2,689/7,762) for AIV. Significant differences in positivity rates were observed in seasons, regions, sampling sites, and specimen types (all p < 0.001). AIV activity peaked in winter and spring, with the highest rates detected in live poultry markets and chopping board swabs. The H9 was the predominant subtype, with co-circulation of multiple subtypes. All 48 H9N2 subtype isolates belonged to the G57 genotype, with the hemagglutinin (HA) and neuraminidase (NA) genes falling into the Y280-like branch, while the internal genes exhibited a mosaic pattern combining G1-like and F/98-like lineages. Molecular characterization analysis revealed multiple mammalian adaptive mutations, involving alterations in receptor-binding sites (T163N, H191N, T197D, T198V, Q234L, Q235M), antigenic epitopes (D280G, N285S), and glycosylation sites (218NRTF, 313NCSK). NA stalk deletion (62–64 aa), along with multiple mutations in the hemadsorption site (E/K368N, D369S/G, D401G/V, N402D, W403L/R, Q432H). Additionally, multiple key amino acid substitutions were also identified in the internal proteins. Conclusion The external environment in western Zhejiang exhibits a high prevalence of AIVs with pronounced spatiotemporal clustering. H9 was the dominant subtype and co-circulated with multiple subtypes, with live poultry markets and slaughterhouses identified as high-risk settings. The H9N2 subtype AIV has accumulated multiple mammalian adaptive mutations, and exhibits genetic linkages across eastern Chinese provinces. These findings collectively underscore the need for an integrated One Health surveillance and early-warning system to reduce the risk of human infections with avian influenza.