Porcine Airway Organoid-Derived Well-Differentiated Epithelial Cultures as a Tool for the Characterization of Swine Influenza a Virus Strains

Swine influenza A viruses (IAVsw) are important causes of disease in pigs but also constitute a public health risk. IAVsw strains show remarkable differences in pathogenicity. We aimed to generate airway organoids from the porcine lower respiratory tract and use these to establish well-differentiated airway epithelial cell (WD-AEC) cultures grown at an air-liquid interface (ALI) for in vitro screening of IAVsw strain virulence. Epithelial cells were isolated from bronchus tissue of juvenile pigs, and airway organoids were cultured in an extracellular matrix in a culture medium containing human growth factors. Single-cell suspensions of these 3D organoids were seeded on Transwell filters and differentiated at ALI to form a pseudostratified epithelium containing ciliated cells, mucus-producing cells and tight junctions. Inoculation with a low dose of IAVsw in a low volume inoculum resulted in virus replication without requiring the addition of trypsin, and was quantified by the detection of viral genome loads in apical washes. Interestingly, inoculation of an H3N2 strain known to cause severe disease in pigs induced a greater reduction in trans-epithelial resistance and more damage to tight junctions than H1N2 or H1N1 strains associated with mild disease in pigs. We conclude that the porcine WD-AEC model is useful in assessing the virulence of IAVsw strains.