The viral ORF3 protein is required for hepatitis E virus apical release and efficient growth in polarized hepatocytes and humanized mice

Hepatitis E virus (HEV), an enterically transmitted RNA virus, is a major cause of acute hepatitis worldwide. Additionally, HEV genotype (gt) 3 can frequently persist in immunocompromised individuals with an increased risk for developing severe liver disease. Currently, no HEV-specific treatment is available. The viral open reading frame 3 (ORF3) protein facilitates HEV egress in vitro and is essential for establishing productive infection in macaques. Thus, ORF3, which is unique to HEV, has the potential to be explored as a target for antiviral therapy. However, significant gaps exist in our understanding of the critical functions of ORF3 in HEV infection in vivo . Here, we utilized a polarized hepatocyte culture model and a human liver chimeric mouse model to dissect the roles of ORF3 in gt3 HEV release and persistent infection. We show that ORF3’s absence substantially decreased HEV replication and virion release from the apical surface but not the basolateral surface of polarized hepatocytes. While the wild-type HEV established a persistent infection in humanized mice, mutant HEV lacking ORF3 (ORF3null) failed to sustain the infection despite transient replication in the liver and was ultimately cleared. Strikingly, mice inoculated with the ORF3null virus displayed no fecal shedding throughout the six-week experiment. Overall, our results demonstrate that ORF3 is required for HEV fecal shedding and persistent infection, providing a rationale for targeting ORF3 as a treatment strategy for HEV infection. Importance HEV infections are associated with significant morbidity and mortality. HEV gt3 additionally can cause persistent infection which can rapidly progress to liver cirrhosis. Currently, no HEV-specific treatments are available. The poorly understood HEV life cycle hampers the development of antivirals for HEV. Here we investigated the role of the viral ORF3 protein in HEV infection in polarized hepatocyte culture and human liver chimeric mice. We found that two major aspects of the HEV life cycle require ORF3: fecal virus shedding and persistent infection. These results provide a rationale for targeting ORF3 to treat HEV infection.

Design and Fabrication of the Vertical-Flow Bioreactor for Compaction Hepatocyte Culture in Drug Testing Application

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.

Assessing the compatibility of primary human hepatocyte culture within porous silk sponges

Porous silk scaffolds hybridized with extracellular matrix proteins are useful for culture of primary human hepatocytes ± supportive non-parenchymal cells.