Development of a real-time quantitative reverse transcriptase PCR assay for detection of the Friend leukemia virus...

Friend leukemia virus (FLV), a murine retrovirus, has been used as a model for elucidation of human immunodeficiency virus (HIV) immunopathogenesis and evaluation of anti-HIV drug effects for several decades. However, no method for direct detection of the plasma viral load has yet been reported. In this study, a TaqMan real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was established for the rapid detection and quantitation of FLV. Measurement of the absolute FLV load was achieved through synthesis of a standard RNA from within the FLV envelope gene for generation of a standard curve. The assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction in a two-step real-time quantitative reverse transcriptase PCR protocol. The relationships between the initially injected FLV dose and the plasma FLV load and spleen index were explored. Following this, the in vivo effects of zidovudine, adefovir dipivoxil, and entecavir on mice infected with FLV were evaluated. The results showed that the plasma FLV load was not proportional to the spleen index over the same FLV injection dosage series, although a trend was observed. When evaluated using plasma viral load, high dose (15 mg/(kg d)) adefovir dipivoxil was capable of significant inhibition of FLV replication in mice. The qRT-PCR assay described here allows specific, sensitive and direct detection of FLV and may also provide more precise measurement of FLV load.