Supporting data for cell culture models for hepatitis E virus (PhD thesis)

Hepatitis E virus (HEV) infection is primarily caused by genotypes within <i>Paslahepevirus balayani</i> (bHEV), although <i>Rocahepevirus ratti</i> (rHEV) has emerged as a zoonoses of concern. Wild-type HEV has a poor record in conventional cell culture, leading to the development of infectious clones that are questionable in terms of truly reflecting natural infection. Most published literature involves highly specific systems for individual variants that have not resulted in clinically translatable findings.In this study, wild-type bHEV and rHEV isolates were successfully propagated in differentiated PLC/PRF/5 cells alongside the gold standard of infectious clone in the HEV field (bHEV^p6). Molnupiravir, ribavirin, and sofosbuvir were shown to have inhibitory effects against wild-type bHEV, but no effect on wild-type rHEV or bHEV^p6. This indicates that infectious clones are not suitable for antiviral studies and that cell culture models capable of better reflecting the immune reaction of host cells to HEV infection are required.The next step was to optimize next-generation models that can act as surrogates for human and rat models. HepaRG^TM cells were shown to propagate HEV isolates, exhibit differential interferon expression over the course of infection, as well as display markers of disease such as increased levels of alanine transaminase and aspartate aminotransferase. Bulk RNA sequencing demonstrated that the impact of bHEV^p6 differs from wild-type HEV isolates in terms of biological pathway activation and gene enrichment pathways. Cryopreserved hepatocytes from both humans and rats were shown to be susceptible to all HEV isolates and require further studies to compare how each host responds to the different HEV variants. Primary rat hepatocytes have also been shown to be capable of supporting infection and antiviral inhibition assays, with the effects of molnupiravir on rat-derived rHEV requiring further investigation. Although rat animal models have been shown to be unsusceptible to bHEV infection, ex vivo rat intestinal tissue has been shown as a promising pilot study into short term infection as it seems to support both bHEV and rHEV culture.Organoids are potentially the best surrogate model to study natural HEV infection. Rat models are important for furthering our knowledge of both human- and rat-derived rHEV while In vivo gerbil models are susceptible to both bHEV and rHEV.This study has conveyed the need for in-depth studies into the more realistic microenvironments generated by primary cells and organoids. The differences between bHEV and rHEV propagation could be due to host and tissue tropism, as well as the nucleotide variation between the species. All models have shown susceptibility to HEV infection to some degree, therefore further investigations into differing impacts of infection between hosts and HEV isolates, such as those performed on HepaRG cells, are required. There are many knowledge gaps in the HEV life cycle and host interaction that have eluded us since it was first discovered, and the development of these models will be key to unraveling the remaining questions around HEV as well as finding more effective therapeutics.