Adaptive selection of quasispecies during <i>in vivo</i> passaging in chickens, mice, and ferrets results in host-specific strains for the H9N2 avian influenza virus

Sporadic human infections of avian influenza virus (AIV) raise significant public health concerns. A critical factor limiting the transmission of AIVs is the shift in receptor-binding preference from Siaα2,3 to Siaα2,6. To reveal the adaptive selection dynamics during the host adaptation process of AIVs, this study generated a viral library with random mutations in the HA gene of the H9N2 strain. Upon passaging the viral library in chickens and mice, the predominantly selected variants exhibited a preference for Siaα2,3 receptors. Notably, the wild-type strain remained dominant in both inoculated and direct-contact chickens, while variants with the ΔL226/R229I substitutions were preferentially selected in mice. Ferrets have a predominance of Siaα2,6 in their respiratory tract. As expected, the variant harboring the N289D mutation, which prefers Siaα2,6 binding, was enriched during <i>in vivo</i> passaging in ferrets. The mice-adapted variant with the ΔL226/R229I mutations causes reduced levels of TNF-α in the early days post-infection in mice, which correlated with an increase in its viral titers. Conversely, elevated levels of IL-6 and IL-1β at five dpi may contribute to the development of the cytokine release syndrome, potentially elucidating the higher fatality rate observed. In conclusion, based on the mutant spectra of the HA gene, this study elucidates the distinct quasispecies dynamics during the adaptation of H9N2 to different hosts, with receptor availability serving as one of the driving factors. Furthermore, a series of critical substitutions that influence the interspecific transmission potential of H9N2 AIVs were identified.IMPORTANCEThe mutation of viruses creates a quasispecies reservoir. In this study, we aimed to investigate the dynamics of quasispecies during the host adaptation of AIVs. We generated a viral library with random mutations in the HA gene of H9N2 and conducted serial passaging in chickens, mice, and ferrets for five generations, respectively. The wild-type strain was dominant in chickens, while mice selected viruses with the ΔL226/R229I substitutions. Both variants showed a preference for binding to Siaα2,3, which aligned with the abundance of Siaα2,3 found in the respiratory tract epithelial cells of chickens and mice. In ferrets, where Siaα2,6 is more prevalent, the variant with the N289D mutation, which prefers Siaα2,6, was found to be enriched. In summary, this study revealed the adaptive selection of H9N2 quasispecies in various hosts, contributing to our understanding of AIV host adaptation.