Abstract
The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of alpha interferon (IFN-α), IFN-λ1, interferon-stimulated gene 15 (ISG15), ISG58, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines.IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development, because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The three-dimensional (3D) porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I and type III interferon (IFN) antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.