QUANTITATION OF FELINE IMMUNODEFICIENCY PROVIRUSES IN DOUBLY INFECTEDCATS 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 immunod eficiency virus (FIV) DNA sequences in the blood of infected cats. Thi s method detects as few as ten copies of a plasmid containing the whol e 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-se nse primer was used in the second amplification step. Amplicons were s ubsequently digested, heat-denatured and loaded on a polyacrylamide ge l in an automated DNA sequencer. The proportion of the two isolates wa s determined using the laser-excited fluorescence of labelled strain s pecific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a com petitive PCR assay, These sensitive and specific assays complement vir ological detection of FIV and enable superinfection studies to be eval uated; a prerequisite for the testing of live attenuated immunodeficie ncy virus vaccines.