The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood. In this study we produced a novel dengue virus type 2 (DV2) subgenomic replicon that expresses a fusion protein comprised of Enhanced Green Fluorescent Protein (EGFP) and Puromycin N-Acetyltransferase (PAC). We successfully established BHK, COS and Huh7 cell lines that stably expressed the DV2 replicon. Using EGFP as a reporter of DV replication complex activity, we set up a new HTS assay. The assay was validated using the inhibitor ribavirin, confirmed by flow cytometry analysis and the analysis of NS5 expression by Western-blot analysis. In order to develop a system to test antivirals against the NS5 proteins of all four DV serotypes in a similar cellular environment, the replicon was further modified, to allow easy exchange of the NS5 gene between DV serotypes. As proof of principle, a chimeric replicon in which the DV2 NS5 gene was substituted with that of DV type 3 was stably expressed in BHK cells and used in ribavirin inhibition studies. The assays described in this study will greatly facilitate DV drug discovery by serving as primary or complementary screening. The approach should be applicable to the development of fluorescent cell-based HTS assays for other flaviviruses, and useful for the study of many aspects of DV, including viral replication and pathogenesis.
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