Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses.
We describe here a novel approach for detecting and quantitating human respiratory syncytial virus (RSV) based on expression of a reporter gene from an RSV minigenome. BHK cells were cytoplasmically transformed with a noncytopathic Sindbis virus replicon expressing T7 RNA polymerase. These cells were then cotransfected with T7 expression plasmids that contain the cDNA of an RSV minigenome and the genes for RSV nucleocapsid proteins N, P, and L. The minigenome contains a reporter gene such as lacZ or CAT flanked by cis-acting RSV transcription signals. Subsequent infection of these cells with RSV resulted in a high level of reporter gene expression which could be inhibited by ribavirin. Mock-infected cells exhibited background levels of expression. This assay can be used to quantitate RSV and titer neutralizing antibody and may be a valuable tool for screening compounds for anti-RSV activity. It serves as a prototype for other negative-strand RNA viruses.
Olivo
P D
PD
Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri, 63110-1093, USA. olivo@borcim.wustl.edu
Collins
P L
PL
Peeples
M E
ME
Schlesinger
S
S
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D016679:Genome, Viral; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D012254:Ribavirin / Q000494:pharmacology; D012845:Sindbis Virus / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D010948:Viral Plaque Assay / Q000379:methods; D014764:Viral Proteins; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics; D005719:gamma-Globulins / Q000276:immunology
D000914:Antibodies, Viral; D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D014764:Viral Proteins; D005719:gamma-Globulins; D012254:Ribavirin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases