Porcine Airway Organoids Reveal Strain-Associated Replication and Epithelial Host Response Signatures of a Reassortant H1N1 Virus

Pigs serve as mixing vessels for influenza A viruses, facilitating reassortment and the emergence of variants with zoonotic potential. Since the 2009 pandemic, human-origin influenza gene segments have been repeatedly detected in swine populations, contributing to the generation of genetically diverse reassortant viruses. However, assessing the infection phenotypes and epithelial host responses of newly emerging reassortant swine influenza viruses remains challenging using conventional in vitro and in vivo approaches. Here, we established long-term expanding three-dimensional porcine airway organoids (pAOs) as a platform for the phenotypic and transcriptomic characterization of reassortant influenza viruses. We compared the infection phenotypes of a recently identified reassortant H1N1 virus, SNU01/H1N1/2023, with those of a classical swine-lineage H1N1 strain, GC0503/H1N1/2005, and the 2009 pandemic strain, CA04/H1N1/2009. All viruses productively infected pAOs, while SNU01 yielded higher levels of infectious progeny than the comparator strains. Bulk transcriptomic profiling at 24 h post-infection revealed that SNU01 infection was associated with an epithelial host-response profile more similar to CA04 than to GC0503, characterized by stronger epithelial antiviral and inflammatory transcriptional activation. Notably, SNU01 infection was associated with distinct cytoskeleton-associated transcriptional programs that were not prominent in the comparator infections. These findings demonstrate that porcine airway organoids can distinguish strain-associated differences in viral replication and epithelial host responses, providing a tractable platform for the comparative characterization of reassortant influenza viruses.