title: Development and characterization of a stable luciferase dengue virus for high-throughput screening.
abstract: To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z' factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Gang
authors.initials: G
authors.lastname: Zou
authors.forename: Hao Ying
authors.initials: HY
authors.lastname: Xu
authors.forename: Min
authors.initials: M
authors.lastname: Qing
authors.forename: Qing-Yin
authors.initials: QY
authors.lastname: Wang
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002213:Capsid / Q000737:chemistry; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D009033:Culicidae; D003716:Dengue Virus / Q000187:drug effects / Q000235:genetics*; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D024882:Drug Resistance, Viral; D017930:Genes, Reporter; D005818:Genetic Engineering; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000235:genetics*; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D008156:Luciferases