Influenza A infection profiling in airway organoids from diverse wildlife and livestock species

This study investigates the susceptibility of diverse wildlife and livestock species to Influenza A viruses using airway organoid cultures. Airway organoids were generated from ten taxonomically distinct species, including mammals and birds, and subsequently differentiated into 2D monolayers to enable controlled viral infection assays. Two Influenza A strains were tested: the 2009 pandemic H1N1 (pH1N1) of swine origin and a clade 2.3.4.4b highly pathogenic avian H5N1 virus. For each species, infection dynamics, cytopathogenicity, and the percentage of infected cells were quantified using high-content imaging and immunofluorescence analysis. Results revealed strong species-specific differences in viral infectivity and host tolerance, which aligned with known in vivo susceptibility patterns for pigs and chickens. Sialic acid receptor profiling showed broadly conserved a2,3- and a2,6-linked receptor distributions but did not fully explain the observed variability in viral behavior. Viral whole-genome sequencing further demonstrated rapid and host-dependent adaptation, including the emergence of mutations in the hemagglutinin receptor-binding domain. Together, these data provide a comparative framework for evaluating influenza host range, viral adaptation, and spillover risk, and position airway organoids as an ethically viable platform for studying viral tropism in wildlife species.