Characterization of an efficientdengue virus replicon for development of assays of discovery of small molecules against dengue virus

Dengue virus (DENV) is apublic health threat to approximately 40% of the global population. At present, neither licensed vaccines nor effective the rapies exist, and the mechanism of viral RNA replication is not well understood. Here, wereport the development of efficient Renilla luciferase reporter-based DENV rep- licons that contain the full-length capsid sequence for transient and stable DENV RNA replication. A com- parison of the transient and stable expression of this RNA-launched replicon to replicons containing various deletions revealed dengue replicon containing entire mature capsid RNA element has higher rep- licon activity. An efficientDNA-lau nched DENV replicon,pCMV-DV2Rep,containing a full-length capsid sequence, was created and successfully applied toevaluate the potency of known DENV in hibitors. Stable cell lines harboring the DENV replicon were easily established by transfecting pCMV-DV2Rep into BHK21 cells. Steady and high replicon reporter signals were observed in the stable DENV replicon cells,even after 30 passages.The stable DENV replicon cells were successfully used to determine the potency of known DENV in hibitors. A high-throughput screening assay based onstable DENV replicon cells was evaluated and shown to have an excellent Z0 factor of 0.74.Altogether, the development of our efficient DENV rep- licon system will facilitate the study of virus replication and the discovery of antiviral compounds.