A human intestinal organoid derived from fetal human colon cells model for studying enteroviral pathogenesis

Human enteroviruses, including enterovirus 71 (EV71), cause hand, foot, and mouth disease (HFMD) and may lead to severe neurological diseases in infants. Enteroviruses first infect the gastrointestinal tract and then spread to the main organs, such as the liver, lungs, heart, and brain. Human intestinal organoids (HIOs) provide a physiologically relevant model for studying enterovirus infections. Unlike traditional two-dimensional (2D) monolayer cultures, HIOs maintain complex epithelial cell diversity and three-dimensional (3D) architecture, allowing for a more accurate representation of in vivo viral-host interactions. In this study, we developed efficient and stable HIOs based on fetal human primary colon cells using the 3D culture system. We discriminated cultured HIOs containing goblet, enteroendocrine, and Paneth cells, and examined the replication efficiency of enteroviruses in HIOs compared to 2D monolayer cultures. HIOs were infected with enteroviruses (EV71, coxsackievirus B3, echovirus 6), and viral replication was assessed using molecular and imaging techniques, which exhibit a higher level of dynamic viral replication in HIOs than in 2D culture. The replication level of enteroviruses increased about 10-fold in HIOs with the virus titre in HIOs was 5-10 times higher than that in 2D cell cultures (p < 0.05). Also, our findings demonstrate that goblet cells serve as a primary site of viral replication. This observation highlights the importance of cellular microenvironments in enteroviral infections and provides insights into gut-specific viral tropism. Collectively, we established an infection model with human intestinal organoids for enteroviruses, providing new opportunities into evaluating enterovirus-related antiviral drugs and modelling enterovirus-associated diseases.