title: Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus.
abstract: Dengue virus (DENV) is a public health threat to approximately 40% of the global population. At present, neither licensed vaccines nor effective therapies exist, and the mechanism of viral RNA replication is not well understood. Here, we report the development of efficient Renilla luciferase reporter-based DENV replicons that contain the full-length capsid sequence for transient and stable DENV RNA replication. A comparison 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 replicon activity. An efficient DNA-launched DENV replicon, pCMV-DV2Rep, containing a full-length capsid sequence, was created and successfully applied to evaluate the potency of known DENV inhibitors. 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 inhibitors. A high-throughput screening assay based on stable DENV replicon cells was evaluated and shown to have an excellent Z' factor of 0.74. Altogether, the development of our efficient DENV replicon system will facilitate the study of virus replication and the discovery of antiviral compounds.
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 350, Taiwan, ROC.
authors.forename: Chi-Chen
authors.initials: CC
authors.lastname: Yang
authors.forename: Ming-Han
authors.initials: MH
authors.lastname: Tsai
authors.forename: Han-Shu
authors.initials: HS
authors.lastname: Hu
authors.forename: Szu-Yuan
authors.initials: SY
authors.lastname: Pu
authors.forename: Ren-Huang
authors.initials: RH
authors.lastname: Wu
authors.forename: Szu-Huei
authors.initials: SH
authors.lastname: Wu
authors.forename: Hui-Mei
authors.initials: HM
authors.lastname: Lin
authors.forename: Jen-Shin
authors.initials: JS
authors.lastname: Song
authors.forename: Yu-Sheng
authors.initials: YS
authors.lastname: Chao
authors.forename: Andrew
authors.initials: A
authors.lastname: Yueh
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D054852:Small Molecule Libraries; D049410:Luciferases, Renilla