Novel replicon-based reporter gene assay for detection of rubella virus in clinical specimens.
Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination.
Tzeng
Wen-Pin
WP
Department of Biology, Georgia State University, 24 Peachtree Center Ave., Atlanta, GA 30303, USA.
Zhou
Yumei
Y
Icenogle
Joseph
J
Frey
Teryl K
TK
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012093:Replicon / Q000235:genetics*; D012409:Rubella / Q000175:diagnosis* / Q000821:virology; D012412:Rubella virus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014709:Vero Cells
D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases