H9N2 avian influenza viruses pose a persistent zoonotic risk owing to their broad host adaptability. Between 2024 and 2025, two H9N2 isolates (ZHY1022B2 and ZHY0417A11) were recovered from quail flocks in Hebei, China. Phylogenetic analysis clustered their HA genes within the dominant B4.7.2 subclade, but diverged into two subgroups (B4.7.2.2 and B4.7.2.1). Of particular interest was ZHY0417A11, which displayed a multigenic reassortment pattern—its PB1 originated from an H3N8 virus, PA and PB2 from H3N3, NS from H10N3, and the M gene was nearly identical (99.32%) to a human H3N8 isolate, whereas the remaining HA, NA and NP segments maintained the H9N2 backbone. Despite the presence of the HA mammalian-adaptive markers (H191N, A198V and Q234L), both strains showed marked antigenic drift from the vaccine strain SS (BJ/94-like; R < 0.5), while retaining reactivity with currently circulating field strains. These findings argue for heightened vigilance in quail populations, given their role as mixing vessels, and highlight the limitations of current vaccine matching in light of ongoing H9N2 evolution.
The H3 subtype avian influenza virus (AIV) poses a substantial global public health threat due to its high host adaptability and ongoing evolution. The recent emergence of novel H3N8 and H3N3 AIVs associated with cross-species transmission underscores the urgent need for enhanced epidemiological surveillance. In this study, we conducted surveillance and characterization of H3 AIVs based on a total of 737 poultry samples collected across 21 Chinese provinces from November 2022 to December 2023. Of these, 69 (9.4%) tested positive for H3 AIV by RT-qPCR, and one H3N8 isolate and ten H3N3 isolates were obtained for whole-genome characterization. We performed whole-genome sequencing, phylogenetic analysis, reassortment inference, and evaluation of key amino acid substitutions, alongside antigenic characterization using hemagglutination inhibition (HI) assays and an in vivo mouse challenge experiment. The H3N8 isolate was identified as a triple-reassortant virus possessing the Eurasian avian H3 gene, the North American avian N8 gene, and H9N2-derived internal genes. The H3N3 isolates represented reassortant viruses that had acquired the HA gene from the novel H3N8 AIV lineage, the NA gene from H10N3 AIV, and internal genes from H9N2 AIV. All isolates exhibited HA cleavage sites characteristic of low pathogenic avian influenza viruses. Additionally, several amino acid substitutions previously associated with enhanced mammalian adaptation were identified, including L89V and I292V in PB2 and H436Y in PB1. In a BALB/c mouse challenge experiment, the representative H3N8 virus established infection without prior adaptation and replicated predominantly in the upper respiratory tract, with detectable viral RNA in respiratory tissues and limited extrapulmonary dissemination. Antigenic analysis revealed no cross-reactivity between the novel H3 AIVs and H5, H7, or H9 AIVs as measured by HI. Based on molecular and phylogenetic characterization, antigenic assessment, and preliminary mammalian infection data, our findings provide evidence suggesting a potential public health risk. We recommend intensified surveillance of H3 AIVs in poultry and accelerated vaccine development to curb viral spread and improve public health preparedness.
Xue Wang, Hao Shi, Peidong Li et al.· The Veterinary Journal· 0 citations
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