title: Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds.
abstract: This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.
authors: Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand. scvlk@mahidol.ac.th
authors: V
authors: V
authors: Leardkamolkarn
authors: W
authors: W
authors: Sirigulpanit
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line / Q000821:virology*; D006224:Cricetinae; D003716:Dengue Virus / Q000235:genetics*; D004353:Drug Evaluation, Preclinical; D047309:Flavones / Q000494:pharmacology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D003698:Hepatitis Delta Virus / Q000235:genetics; D017874:Immediate-Early Proteins / Q000235:genetics; D007769:Lactams / Q000494:pharmacology; D016712:Mupirocin / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics
chemical_list: C501188:5,6,2',3',6'-pentamethoxyflavone; D000956:Antigens, Viral; D000998:Antiviral Agents; D047309:Flavones; D017874:Immediate-Early Proteins; D007769:Lactams; D016337:RNA, Catalytic; D012367:RNA, Viral; D014764:Viral Proteins; C044582:immediate-early proteins, cytomegalovirus; C107034:thiomarinol F; D049452:Green Fluorescent Proteins; D016712:Mupirocin