Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the leading causes of acute viral hepatitis in develop-ing countries. Historically, as both viruses have been thought to only cause acute disease, interest in study-ing them has been low. As a result, the pathogenesis of infection and the immune responses generated by HAV and HEV infection remain poorly understood. Despite the absence of viral glycoproteins, both viruses acquire a host cell-derived lipid envelope during virus morphogenesis, allowing the virus to bud in a non-cytolytic fashion. This quasi-envelope protects HAV and HEV from neutralizing antibodies and facilitates systemic spread within the host. The non-enveloped virions, which are shed in feces, are very stable and allow for epidemic transmission. Both viruses spread by the fecal-oral route in humans through contaminat-ed water and food. The intestinal epithelium is the first physical barrier that HAV and HEV must cross to enter the bloodstream and gain access to hepatocytes, their major replication site. During the natural spreading of HAV and HEV along the gut-liver axis, viruses must enter intestinal epithelial cells by their api-cal side and de novo viruses have to be released from their basolateral side en route to the liver. Once in the liver, these viruses will infect hepatocytes from their basolateral side and de novo virus particles will be re-leased back into the gastro-intestinal tract by polarized apical secretion into the bile canaliculi. We found that the polarized nature of intestinal epithelial cells strongly influences host/pathogen interactions. Precise-ly, the side of infection by viruses (apical vs. basolateral) strongly influences the intrinsic immune response generated by intestinal epithelial cells (Stanifer et al., 2019). In addition, secretion of cytokines by these cells also occurs in a polarized manner (Stanifer et al. 2016; collaboration Binder, Bartenschlager). As such, to fully characterize the life cycles of HAV and HEV, it is critical to integrate spreading of these viruses along the gut/liver axis and to consider the polarized nature of both intestinal epithelial cells and hepato-cytes.
In this proposal, the long term aims are to address how the enteric phase of the HAV and HEV life cycle impacts virus replication and spread in the liver and ultimately whether this contributes to the acute nature of HAV and HEV infection. To this end, we will exploit both human mini-gut organoids and stem cell-derived hepatocytes to study HAV and HEV infection, replication, and spread using primary, non-transformed human cells. Together with Lohmann, Binder, Pichlmair, Bartenschlager, and Z03N, we will:
1.) Study the impact of cellular polarity on HEV infection and immune response in human intestinal epithelial cells (IECs). Here, we will exploit both immortalized lines and non-transformed IECs grown as organoids to study the importance of interferon in controlling HEV infection in human intestinal epithelial cells. Using organoids, we will reproduce the polarized and multi-cellular nature of the gut epithelium and characterize host/HEV interaction in a cell type-specific manner.
2.) Characterize the innate immune response generated by human hepatocytes upon HAV and HEV infection and the impact of cellular polarity. In this aim we will study the response by HAV and HEV infec-tions in immune competent hepatocyte culture systems. We will further analyze if the innate immune pro-gram is polarized, similar to what has been observed in other epithelial cells.
3.) Model and characterize the impact of the primary enteric infection on HAV and HEV infection of hepatocytes. Here, we want to address whether IEC-derived viral particles and/or IEC-derived molecules can impact the hepatic phase of HAV/HEV infection.