title: A rapid and sensitive real-time PCR assay for the K65R drug resistance mutation in SIV reverse transcriptase.
abstract: 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.
authors.affiliation: National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia 30303, USA. jjohnson@cdc.gov
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Johnson
authors.forename: Koen K A Van
authors.initials: KK
authors.lastname: Rompay
authors.forename: Eric
authors.initials: E
authors.lastname: Delwart
authors.forename: Walid
authors.initials: W
authors.lastname: Heneine
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D004195:Disease Models, Animal; D024882:Drug Resistance, Viral / Q000235:genetics*; D008251:Macaca / Q000821:virology; D008826:Microbial Sensitivity Tests / Q000379:methods; D063065:Organophosphonates / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D015302:Simian Immunodeficiency Virus / Q000187:drug effects / Q000235:genetics*; D000068698:Tenofovir
chemical_list: D063065:Organophosphonates; D018894:Reverse Transcriptase Inhibitors; D000068698:Tenofovir; D012194:RNA-Directed DNA Polymerase; D000225:Adenine