Cloning full-length env gene from plasma viral RNA

To clone the env gene from the extracted plasma viral RNA, cDNA was first synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR with primers 07Rev8 and 1.R3.B3R ( Table S1 ). The full-length env gene was then amplified using primers HZBOB and HZBCOE for the first-round PCR, and primers HZBIB and HZBCIE for the second-round PCR. Full-length env nucleotide sequences were cloned into pcDNA3.1-TOPO-V5-His vector (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The plasmids were analyzed by restriction analysis (Hind III and XbaI) to confirm the presence of the insert.