Ultrasensitive allele-specific PCR reveals rare preexisting drug-resistant variants and a large replicating virus population in macaques infected with a simian immunodeficiency virus containing human immunodeficiency virus reverse transcriptase

Abstract

It has been proposed that most drug-resistant mutants, resulting from a single-nucleotide change, exist at low frequency in human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) populations in vivo prior to the initiation of antiretroviral therapy (ART). To test this hypothesis and to investigate the emergence of resistant mutants with drug selection, we developed a new ultrasensitive allele-specific PCR (UsASP) assay, which can detect drug resistance mutations at a frequency of ≥0.001% of the virus population. We applied this assay to plasma samples obtained from macaques infected with an SIV variant containing HIV-1 reverse transcriptase (RT) (RT-simian-human immunodeficiency [SHIV](mne)), before and after they were exposed to a short course of efavirenz (EFV) monotherapy. We detected RT inhibitor (RTI) resistance mutations K65R and M184I but not K103N in 2 of 2 RT-SHIV-infected macaques prior to EFV exposure. After three doses over 4 days of EFV monotherapy, 103N mutations (AAC and AAT) rapidly emerged and increased in the population to levels of ∼20%, indicating that they were present prior to EFV exposure. The rapid increase of 103N mutations from <0.001% to 20% of the viral population indicates that the replicating virus population size in RT-SHIV-infected macaques must be 10(6) or more infected cells per replication cycle.

Fig 1

Plasma viral RNA shown for two pigtail macaques infected with RT-SHIV_mne at week 0 and viremia monitored by RT-PCR. Circled symbols indicate time points measured in this study for each animal. After plasma was collected at week 13, three doses of EFV monotherapy were administered over 4 days as indicated by the bracket. Daily cART containing TNV, FTC, and EFV was initiated at week 17. Adapted from Fig. 2A in reference .

Fig 2

Mutant copy number versus RNA copy number/ml of plasma virus in the treated RT-SHIV-infected macaques, M03250 (A) and M04008 (B). The frequencies of the indicated mutations were assessed by UsASP. Open squares with downward arrows indicate time points where the mutant copy number was less than the sensitivity of the assay.