Rapid and convenient assays to assess potential inhibitory activity on in vitro hepatitis A replication

• We established three assays to screen for hepatitis A virus replication inhibitors.
• Assays were validated with known inhibitors interferon-alpha and amantadine HCl.
• The protease inhibitor rupintrivir displays strain-dependent activity.
• The broad-spectrum enterovirus inhibitor enviroxime is devoid of activity.

Three different antiviral assays were developed for the in vitro screening of inhibitors of the hepatitis A virus (HAV) of which (i) a cytopathic effect reduction assay suitable for medium-to-high-throughput screening and (ii) two virus yield reduction assays (based on quantification of viral RNA) for genotypes IB and IIIA. The assays were validated for antiviral studies with interferon-alpha (IFNα) and amantadine HCl, two known inhibitors of HAV replication. IFNα effectively inhibited HAV replication, whereas the activity of amantadine HCl appeared to be strain-dependent. Employing these assays, we assessed the effect of the known enterovirus inhibitors pleconaril, rupintrivir and enviroxime on HAV replication. Pleconaril exhibited some very moderate activity, the effect of rupintrivir proved to be strain-dependent. Enviroxime did not inhibit HAV replication, suggesting that phosphatidylinositol-4-kinase IIIβ is not crucial in the HAV life cycle.