Quantitation of feline immunodeficiency proviruses in doubly infected cats using competitive PCR and a fluorescence-based RFLP

A nested polymerase chain reaction assay, which amplifies a region of the gag gene, was developed for the direct detection of feline immunodeficiency virus (FIV) DNA sequences in the blood of infected cats. This method detects as few as ten copies of a plasmid containing the whole genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV isolates in double infected cats: we devised an RFLP analysis on PCR amplified products exploiting sequence differences in the gag gene of the two strains. To quantitate the two strains, a fluorescent inner-sense primer was used in the second amplification step. Amplicons were subsequently digested, heat-denatured and loaded on a polyacrylamide gel in an automated DNA sequencer. The proportion of the two isolates was determined using the laser-excited fluorescence of labelled strain specific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a competitive PCR assay, These sensitive and specific assays complement virological detection of FIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.