Characterization of chicken epithelial cell types and their manipulation to investigate avian influenza virus infection
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[EMBARGOED UNTIL 08/01/2026] Understanding the diversity, function, and susceptibility of avian respiratory epithelial cells is essential for advancing research on host--pathogen interactions and respiratory disease in poultry. To address the current lack of physiologically relevant models, this work combines in vitro and in vivo approaches to characterize epithelial cell identity, behavior, and response to infection. A primary chicken tracheal epithelial cell culture system was established and optimized to support the long-term growth and maintenance of epithelial characteristics (Chapter 2). Media formulations were identified that preserved epithelial morphology and expression of airway markers while others promoted differentiation into specialized cell types. Inhibition of BMP and ROCK signaling pathways enhanced proliferation and extended culture longevity with treated cells retaining key epithelial features. Transcriptomic profiling revealed active BMP signaling in untreated cells, which was suppressed upon inhibitor treatment, supporting the effectiveness of this approach. These cultures remained susceptible to infection by different strains of avian influenza viruses. To build upon these findings in a tissue context, single-cell RNA sequencing was used to construct a high-resolution atlas of non-immune cell populations in the chicken lung (Chapter 3). Four major epithelial subtypes--alveolar type 1, alveolar type 2, basal, and supporting cells--were identified based on distinct transcriptional profiles. Cell--cell communication analysis revealed extensive signaling networks, particularly involving AT1 cells, and pseudotime analysis suggested lineage progression from basal to alveolar cells. Spatial mapping by multiplex in situ hybridization confirmed the localization of these epithelial populations within alveolar structures. Together, these studies provide foundational insights and tools for investigating avian epithelial biology, epithelial--viral interactions, and future gene editing applications.
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M.S.