Development and characterization of chicken lung organoids for in vitro modeling of avian influenza virus-host cell interaction

High pathogenicity avian influenza viruses pose a growing threat to poultry, livestock, wildlife, and humans as they undergo accelerated expansion of host and geographical ranges. Since 2020, these viruses have driven a panzootic characterized by extensive viral diversification and spillover into species previously considered to be resistant. There is currently a lack of physiologically relevant in vitro models that can be used to screen the rapidly changing viral landscape. To address this need, we describe the first chicken lung organoids derived from adult stem cells of specific pathogen free White Leghorns. We analyze their gene expression with bulk RNA sequencing, confirm their cellular heterogeneity via single-nuclei RNA sequencing, and provide basic morphological characterization using hematoxylin and eosin staining, immunohistochemistry, and transmission electron microscopy. The results indicate that the organoids contained several cell types, including non-ciliated columnar, cuboidal, squamous, and mucin-producing cells, representative of different regions of the avian respiratory system. Furthermore, expression of genes relevant to influenza A virus infection and replication appeared to be conserved across organoid and tissue samples. Infections revealed that chicken lung organoids support robust replication of both low and high pathogenicity avian influenza A viruses, with high pathogenicity strains showing more rapid amplification. Therefore, these organoids have the potential to effectively model viral infection, enabling the investigation of viral pathogenesis and evolutionary potential, virus-host interactions, and antiviral targets.