Multi-section surveillance of AIVs in poultry exposure environments and hospitalized PUE in three Chinese cities

The global expansion of avian influenza viruses (AIVs), particularly clade 2.3.4.4b H5 viruses, continues to pose a significant zoonotic threat. Although Chinese vaccination-plus-culling strategy has reduced H5- and H7-exposure disease activity, continued viral evolution and reassortment highlight the need for sustained surveillance at the human-environment interface. Environmental samples from live poultry markets (LPMs), slaughterhouses and poultry farms/backyards, together with respiratory specimens from hospitalized patients with pneumonia of unknown etiology (PUE) were collected in three Chinese cities from December 2021 to December 2023. Specimens were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR) for influenza A virus (IAV), H5, H7, H9 and H10 subtypes. Positive rates among cities and sampling sites were compared using chi-square tests. Monthly positive rates among environmental sample categories were compared using the Kruskal-Wallis test, and paired monthly rates between 2022 and 2023 using the two-sided Wilcoxon signed-rank test. H5, H9 and H10 viruses underwent HA gene sequencing and phylogenetic analysis. Among 8,335 environmental samples, the overall IAV positive rate was 32.2%; H9 predominated (20.1%), followed by H5 (7.1%), while H10 was sporadic (0.2%) and H7 was not detected. H5 and H9 positive rates were higher during autumn and winter. Median monthly positive rates of H5 and H9 were lower in 2023 than in 2022, although without significant differences. H5 and H9 positive rates were generally higher in LPMs and slaughterhouses than in farms/backyards. Among environmental sample categories, H5 showed no significant difference ( P = 0.06), whereas H9 differed significantly ( P = 0.03), with higher median rates in cutting boards and blood sewage than in drinking water. Phylogenetically, all H5, H9, and H10 viruses belonged to the Eurasian lineage, with H5 viruses clustering in clade 2.3.4.4b. Several environmental H9 viruses were closely related to representative human H9N2 viruses. Of 840 PUE respiratory specimens, 86 were positive for seasonal influenza viruses, but none for H5, H7, H9 or H10. Multiple AIV subtypes continued to circulate in poultry-exposure environments. Sustained multi-site One Health surveillance integrating poultry-exposure environments and human populations is warranted to monitor AIV evolution and zoonotic risks.