Monitoring of Hepatitis E Virus Infection and Replication by Functional Tagging of the ORF2 Protein

Background and Aims: Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems.

Methods: We exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV ORF2 protein, corresponding to the viral capsid, allowing the insertion of reporter sequences in a functional context.

Results: Short sequence insertions (5 aa) were tolerated at four distinct sites in the C-terminal region of ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, while with about 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies.

Conclusions: We describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture.