From each plasma specimen, ∼20,000 viral RNA copies were extracted using the QIAamp Viral RNA Mini kit (QIAGEN). RNA was eluted and immediately subjected to cDNA synthesis. Reverse transcription of RNA to single-stranded cDNA was performed using SuperScript III reverse transcription according to manufacturer’s recommendations (Invitrogen). In brief, each cDNA reaction included 1× RT buffer, 0.5 mM of each deoxynucleoside triphosphate, 5 mM dithiothreitol, 2 U/ml RNaseOUT (RNase inhibitor), 10 U/ml of SuperScript III reverse transcription, and 0.25 mM antisense primer SIVsm/macEnvR1 5′-TGTAATAAATCCCTTCCAGTCCCCCC-3′ (nt 9454–9479 in SIVmac239). The mixture was incubated at 50°C for 60 min, followed by an increase in temperature to 55°C for an additional 60 min. The reaction was then heat-inactivated at 70°C for 15 min and treated with 2 U of RNase H at 37°C for 20 min. The newly synthesized cDNA was used immediately or frozen at −80°C.