Abstract
Background and AimsHepatitis 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. MethodsWe 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. ResultsShort 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. ConclusionsWe describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact And ImplicationsHepatitis E virus (HEV) infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing to evaluate antiviral drugs and neutralizing antibodies.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Similar Papers
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.