Proliferative cell targeting and epithelial cell turnover fuels hepatitis E virus replication in human intestinal enteroids

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). Three strategies were explored to establish an HEV-HIE model: three-dimensional (3D) HIEs, two-dimensional (2D) 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. HEV infection in 3D-HIEs and 2D-HIEs showed limited replication, whereas HIEs electroporation led to a sustained increase in the release of non-enveloped infectious virions. These virions could re-infect new 3D-HIEs, yielding a [~]2 log10 increase in HEV RNA. In electroporated HIEs, high expression of the infectious ORF2 capsid form was observed in the supernatant. Importantly, 70% of all HEV-infected cells were identified as proliferative cells. ORF2 staining was also observed in absorptive enterocytes, goblet, and enteroendocrine cells. Overall, we established a robust HEV-HIE model that yields high titers of infectious non-enveloped 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 non-enveloped HEV shed in the feces. SignificanceHepatitis E virus (HEV) causes [~]20 million enterically transmitted cases of viral hepatitis globally. To better understand the largely unexplored role of the gut during an HEV infection, we used human intestinal enteroids (HIEs). We show that HEV-electroporated HIEs produce highly infectious non-envelopedHEV particles, resulting in a more efficient re-infection compared with traditional gold-standard methods. Importantly, we detected HEV in multiple human intestinal epithelial cell types within the human intestinal epithelium, with the majority of proliferative cells infected. This model offers valuable insights into the intestinal tropism of HEV and can serve to further investigate chronic HEV infection in the gut. Moreover, this underscores the importance of using physiologically relevant models to unravel details of HEV infection in the gut.