Modeling reptile virus infection in vitro using Python regius airway organoids.

Zoonoses pose substantial global health risks, highlighting the need to better understand animal-to-human transmission. Reptiles are increasingly recognized as hosts of diverse pathogens, including numerous viruses, yet the diversity and prevalence of reptile pathogens, as well as their potential risk to humans, remain poorly understood. Here, we establish and characterize airway organoids derived from Python regius, providing an in vitro model to study reptile airway infection. Through de novo assembly of a Python regius reference genome, we characterize airway organoids at single-cell resolution, which suggests the presence of diverse cell populations including ionocytes, ciliated, secretory, goblet, endocrine, tuft, and basal cells. The organoids support productive infection with Ball Python Nidovirus (BPNV) and mount a robust epithelial antiviral response through the induction of interferon-stimulated genes, cytokines, and genes involved in chemical defense. As a proof-of-concept, treating organoids with antiviral drugs during infection reduces BPNV levels, highlighting the model's utility for drug testing. By providing a reductionist system of the serpentes airway, these organoids constitute a physiologically relevant in vitro model to study reptile viruses and host-pathogen interactions in their native host.