Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Abstract

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fuorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fuorescent dye, which permits the specifc quantifcation and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fuorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identifcation of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by diferent RdRps, illustrated here using purifed RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fuorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.