Proliferative Cell Targeting and Epithelial Cell Turnover Fuels Hepatitis E Virus Replication in Human Intestinal Enteroids.

Background and Aims: Hepatitis E virus (HEV) is a leading pathogen causing acute viral hepatitis globally. While HEV is primarily spread fecal-orally, the role of the gut in HEV pathogenesis remains largely unexplored, including how HEV disseminates from gut to liver, and whether the gut is an HEV reservoir. We here aimed to illuminate HEV biology in the gut using human intestinal enteroids (HIEs). Methods: Three strategies were explored to establish an HEV-HIE model - three-dimensional (3D) HIEs, two-dimensional HIEs in transwell, and HEV RNA-electroporated HIEs. HEV particles produced by electroporated HIEs were characterized by western blot and gradient centrifugation. The intestinal tropism of HEV was investigated through confocal fluorescent microscopy and gene expression analysis. Results: HEV infection in 3D-HIEs and two-dimensional-HIEs showed limited replication, whereas HIEs electroporation led to a sustained increase in the release of nonenveloped infectious virions. These virions could reinfect new 3D-HIEs, yielding a ∼2 log10 increase in HEV RNA over time. In electroporated HIEs, high expression of the infectious open reading frame 2 capsid form was observed in the supernatant. Importantly, 70% of all HEV-infected cells were identified as proliferative cells (leucine-rich-repeat-containing G-protein-coupled receptor 5 intestinal stem cells and transit-amplifying progenitor cells). Open reading frame 2 staining was also observed in absorptive enterocytes, goblet, and enteroendocrine cells. Conclusion: Overall, we established a robust HEV-HIE model that yields high titers of infectious nonenveloped virions. Proliferative cells and the fast intestinal epithelial cell turnover are important features that facilitate efficient HEV replication, and likely also its dissemination. This study suggests that the gut is an HEV reservoir, capable of producing some of the nonenveloped HEV shed in the feces.