Background: Avian influenza virus (AIV), a zoonotic pathogen found worldwide, includes multiple subtypes, one of which is the H4 subtype frequently detected in wild birds and poultry. Despite its prevalence, research on H4 subtype AIV has been scarce, with a focus predominantly on the H4N2 and H4N6 subtypes. The zoonotic potential of H4N1 has not been investigated to date.

Methods: In this study, we used gene sequencing in conjunction with bioinformatics methodologies to analyze wild-type H4N1 AIV strain and mutant strains emerging from serial passaging in cell culture. Furthermore, we assessed the zoonotic potential of H4N1 and the alterations caused by mutations via a series of phenotype assays, including evaluation of receptor binding affinity, immunofluorescence assays, analyses of growth kinetics across different animal cell cultures, and in vivo pathogenicity studies.

Results: Our research reveals that H4N1 AIV can bind to human receptors and exhibits an affinity for human lung and tracheal tissues. In vitro experiments demonstrate that H4N1 replicates efficiently in human cell lines. Furthermore, animal studies demonstrate that H4N1 can induce pneumonia in mice without the need for prior adaptation to the host. Notably, during passage in cell culture, H4N1 rapidly acquired two previously unreported mutations. These mutations significantly enhanced the virus´s ability to attach to human receptors and its capacity for replication.

Conclusions: In summary, our study provides preliminary experimental evidence for the emerging zoonotic potential of H4N1 AIV. These findings expand our knowledge of the H4 subtype AIV and reinforce the critical need for continued surveillance of AIV to prevent and prepare for potential outbreaks affecting human health.