COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION FOR QUANTIFYING MURINE AIDS VIRUS

The causative agent of murine AIDS (MAIDS) is the defective murine leu kemia virus BM5d, that requires the replication-competent ecotropic Mu LV (BM5e) helper virus. We developed a competitive quantitative PCR me thod including specific internal standards to quantify, the expression of BM5d in the spleen of infected mice and to characterize BM5d expre ssion kinetics following experimental infection. Specimen RNA was reve rse-transcribed and co-amplified with a competitive template containin g a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restricti on site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defectiv e-virus mRNA was compared to a constant copy number of murine beta acr in mRNA. LP-BM5 production was measured in the spleen of infected mice . Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicat ive virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.