Hepatitis E virus replication is facilitated by epithelial cell turnover and targets enteroendocrine cells in human intestinal organoids

Hepatitis E virus (HEV) is an emerging pathogen responsible for acute viral hepatitis globally. There is no specific antiviral and non-universal vaccine, partly due to limitations in its in vitro cultivation. Although HEV is mainly fecal-oral transmitted and excreted in the feces in high titers, the role of the gut in HEV-induced disease remains unexplored. Little is known about how the virus spreads from gut to liver, or about the gut potentially acting as an HEV reservoir. To address this, we developed an HEV infection model using human intestinal enteroids (HIEs). HEV infection was done using: a) differentiated 3D-HIEs; b) differentiated 2D-HIEs in transwells; c) electroporation of HIEs with HEV capped RNA. 3D infection with HEV-3 strains resulted in a limited increase in HEV RNA. Infection of 2D-HIEs yielded similar replication levels, with shedding mainly to the apical side of the intestinal epithelial layer. Importantly, electroporation of HIEs with HEV-3 RNA resulted in a sustained increase in viral load over time in the supernatant, with the detection of infectious virus particles. Likewise, electroporation of HEV-1 and 3 luciferase replicon RNA, a 5-fold and 20-fold increase in luciferase signal was observed. Treatment with the current off-label ribavirin effectively inhibited viral replication in the luciferase subgenomic or full-length genomic HEV RNA electroporated HIEs.

Moreover, here we investigate the cellular tropism of hepatitis E virus in HIEs. Abundant HEV ORF2 antigen was detected in infected HIEs, including proliferating cells, enterocytes and enteroendocrine cells, with increased mRNA expression for the latter cell type. Overall, we here established a model for HEV infection in HIEs and discovered that the fast cell turnover of the intestine is an important feature that enables efficient HEV replication. Furthermore, HEV infection promotes differentiation towards enteroendocrine cells, which the virus can infect.