2021 Fiscal Year Annual Research Report
Production of infectious hepatitis E virus (HEV) harboring bioluminescent reporter gene for comprehensive screening of antiviral drugs against HEV and ex vivo evaluation of selected drugs
| Project/Area Number |
20K22771
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| Research Institution | Jichi Medical University |
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Principal Investigator |
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| Project Period (FY) |
2020-09-11 – 2022-03-31
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| Keywords | hepatitis E virus / reporter virus / nanoKAZ / hypervariable region / drug screening / quasi-enveloped HEV / non-enveloped HEV |
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| Outline of Annual Research Achievements |
Hepatitis E virus (HEV) is a quasi-enveloped virus with single-stranded positive-sense RNA genome. Studies of molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, stability of large foreign genes is difficult to maintain after insertion into viral genome. Ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, development of novel anti-HEV drugs is of great importance.
We developed a system consisting of recombinant infectious HEV harboring small luciferase gene (nanoKAZ) in hypervariable region (HVR) of open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had similar morphological characteristics to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious.
HEV particles circulating in bloodstream and attaching to hepatocytes in infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. It covers at least inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified and confirmed to be effective in cultured cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants and ORF2 protein expression intracellularly, thus may be candidate novel anti-HEV drugs.
The results of this study provide evidence supporting the use of this system in more variable HEV studies.
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