Isolation and Genetic Characteristics of Novel H4N1 Avian Influenza Viruses in ChongQing, China

Background: Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent call for vigilance. Methods: We developed a comprehensive protocol that incorporates nucleic acid testing, gene sequencing, isolation culture, and resequencing for the evaluation of AIV in environmental samples. During this process, the H4N1 subtype of AIV isolated from a live poultry market was subjected to whole-genome sequencing and subsequent analysis. Results: Between December 2021 and November 2023, 934 environmental samples were evaluated, and we isolated an H4N1 subtype AIV (CS01) for the first time from a live poultry market and determined its complete genome sequence. Subsequent analysis revealed that CS01 shares genetic characteristics with H4N2, H6N1, and H3N2; moreover, the receptor-binding pocket sequence of CS01 exhibited high similarity to that of other H4N1 strains, with 32 notable mutation sites. Conclusions: Our study heralded the first isolation of the H4N1 subtype of AIV from a live poultry market teeming human activity. Subsequent analysis revealed that CS01 is a recombinant virus that possesses zoonotic potential and harbors a variety of pathogenicity-enhancing mutations. The isolation of CS01 highlights the importance of active monitoring in heavily contaminated live poultry markets as an effective approach to study AIVs.