Rapid and sensitive real-time PCR assay for the K65R drug resistance mutation in SIV reverse transcriptase

Macaques infected with simian immunodeficiency virus (SIV) provide a suitable model for assessing the efficacy of antiretroviral (ARV) drug interventions and drug resistance selection associated with treatment. Resistance to the HIV reverse transcriptase inhibitor tenofovir continues to be examined in different treatment strategies in the macaque model. Evaluations of treatment interventions and drug resistance are hampered by the limited sensitivity of conventional population sequencing and the substantial effort involved in testing various tissue compartments in which viruses may reside. Therefore, a sensitive assay that permits simple and rapid testing for drug-resistant viruses would benefit appraisals of ARV treatments using in vivo models. To have this capability, we developed a real-time PCR-based assay for the detection of the SIV K65R reverse transcriptase mutation, a key marker for reduced susceptibility to tenofovir. Evaluations of SIV sequences yielded an assay detection limit mean of 0.4% mutant virus (range = 0.1 - 2%) in a wild-type background. In testing longitudinal plasma specimens from four SIV-infected macaques that received an active daily regimen of 30 mg/kg of tenofovir subcutaneously, the assay was able to detect K65R-positive viruses in all animals within 1 - 7 weeks after treatment began. The emerging mutants were initially present at frequencies estimated between 0.4% and 3%, below the detection capability of population sequencing. We propose the SIV K65R real-time PCR assay provides improved sensitivity and simplicity in studying tenofovir resistance in macaque models.