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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 fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, <jats:italic>de novo<\/jats:italic> RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.<\/jats:p>","DOI":"10.1038\/s41598-019-41998-1","type":"journal-article","created":{"date-parts":[[2019,4,1]],"date-time":"2019-04-01T11:29:55Z","timestamp":1554118195000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":53,"title":["Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs"],"prefix":"10.1038","volume":"9","author":[{"given":"Yanira","family":"S\u00e1ez-\u00c1lvarez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4138-4608","authenticated-orcid":false,"given":"Armando","family":"Arias","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"del \u00c1guila","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rub\u00e9n","family":"Agudo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,4,1]]},"reference":[{"key":"41998_CR1","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1128\/JVI.72.1.73-83.1998","volume":"72","author":"G Kuno","year":"1998","unstructured":"Kuno, G., Chang, G. 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