Professor of Comparative Medicine University of Georgia Athens, Georgia, United States
Abstract: Avian influenza viruses typically reside in wild waterfowl, inducing no clinical disease. When these low pathogenic strains spill over to domestic poultry, they evolve into highly pathogenic strains (HPAIV), with devastating effects on the industry. Spillover events have been increasingly reported in mammals (including humans), causing severe disease with high mortality. There is, therefore, a critical need to understand the viral tropism, host immune responses, and replication dynamics of HPAIVs in various avian species. 2D cell cultures, which have been used to study HPAIVs, have shortcomings, including their lack of cellular heterogeneity. 3D organoids, composed of multiple cell types, may represent a more faithful model to study mechanisms of HPAIV dynamics and pathogenicity. Adult stem cell-derived lung organoids from three-week old SPF White Leghorn chickens were developed and their transcriptome analyzed via bulk and single-nuclei RNA sequencing. Morphology was characterized using brightfield imaging, H&E, immunohistochemical (IHC), immunofluorescent, and special staining in tissues and organoids. Three organoid lines were established, passaged (n >10), and cryopreserved. Histological staining revealed columnar, cuboidal, squamous, and acidic mucin-producing cells, representing regions throughout the lung. Single-nuclei RNA sequencing identified epithelial and fibroblast cell populations in the culture, confirmed with IHC. Epithelial cells were further resolved to be either cycling or non-cycling. Bulk RNA sequencing revealed highly conserved gene expression between organoids and parent tissues. Furthermore, similar expressions (in TPM) of both lung-relevant and virus-related genes were noted. This study established the first chicken lung organoids derived from adult stem cells. RNA and protein expression in the avian lung organoids and parent tissues suggest organoids can be used to model epithelial cell populations in birds for HPAIV studies.
