Development of a new comprehensive HIV-1 genotypic drug resistance assay for all commercially available reverse transcriptase, protease and integrase inhibitors in patients infected with group M HIV-1 strains

Characterization of a novel HIV-1 circulating recombinant form, CRF91_cpx, comprising CRF02_AG, G, J, and U, mostly among men who have sex with men

“…The blood samples were processed within two hours of sampling at the Laboratory of Biotechnology and Molecular Virology. The isolation of plasma from whole blood and subsequent HIV-1 RNA extraction from plasma using the QIAamp Viral RNA Mini Kit (QIAGEN, 52904) were performed according to previously described methodology and the manufacturer’s instructions [ 17 ]. As of 20 May 2021, blood samples from 277 HIV-1-infected patients had been collected as part of the prospective molecular epidemiology study (C. Topcu et al ., manuscript in preparation for publication) conducted from 9 March 2017 to 14 October 2021.…”

“…As of 20 May 2021, blood samples from 277 HIV-1-infected patients had been collected as part of the prospective molecular epidemiology study (C. Topcu et al ., manuscript in preparation for publication) conducted from 9 March 2017 to 14 October 2021. The pol ( protease, reverse transcriptase, integrase and partial vif ) region (2253–5250 in the HXB2 genome) sequences of the 269 HIV-1 viral genomes isolated from the 277 blood samples were successfully amplified according to a previously reported touchdown RT–PCR protocol, while the remaining eight samples were PCR negative [ 17 ]. The SuperScript™ IV One-Step RT–PCR System (ThermoFisher Scientific, 12594025) was used for primary RT–PCR, and Platinum™ Hot Start PCR 2× Master Mix (ThermoFisher Scientific, 13000012) was used for secondary nested PCR.…”

“…A positive RNA control and a negative control were used in all PCR amplifications to confirm the success of the experiments and rule out the possibility of contamination, respectively. The sequencing of the 3259 bp final product of the pol RT–PCR assay employing 10 sequencing primers was conducted through Sanger sequencing by Macrogen Europe ( https://dna.macrogen-europe.com/eng/ ) [ 17 ]. Finally, the HIV-1 genotypic subtypes based on the pol region nucleotide sequences were determined using the REGA HIV-1 Subtyping Tool Version 3.0 [ 18 ].…”

“…With the aim of identifying a possible newly emerging circulating recombinant form, near full-length HIV-1 genomes (790–8795 in the HXB2 genome) of the 14 HIV-1 recombinants encoding the gag, pol, vif, vpr, tat, rev, vpu, env and partial nef regions were amplified and sequenced using an in-house-developed near full-length HIV-1 genome RT–PCR assay (C. Topcu et al ., manuscript in preparation for publication). This assay comprises three overlapping amplicons spanning the whole genome amplified via a previously described pol RT–PCR assay (2253–5250 in the HXB2 genome) [ 17 ], gag RT–PCR assay (790–2292 in the HXB2 genome) and env RT–PCR assay (5041–8795 in the HXB2 genome). The three PCR assays utilized HIV-1-specific primers targeting various HIV-1 group M subtypes, CRFs and recombinants.…”

“…When all amplicons obtained with the sequencing primers were composed, the mean number of aligned nucleotides per position was found to be approximately three, ranging from one to six at each nucleotide position. The quality of the sequencing readout and the success of each sequencing primer amplicon were defined as previously published [ 17 ]. Subsequently, the final HIV-1 gag and env region amplicons were aligned with the previously obtained HIV-1 pol region amplicons to obtain the consensus nucleotide sequences.…”