Single genome sequencing of near full-length HIV-1 RNA using a limiting dilution approach

Sequencing very long stretches of the HIV-1 genome can advance studies on virus evolution and in vivo recombination but remains technically challenging. We developed an efficient procedure to sequence near full-length HIV-1 RNA using a two-amplicon approach. The whole genome was successfully amplified for 107 (88%) of 121 plasma samples including samples from patients infected with HIV-1 subtype A1, B, C, D, F1, G, H, CRF01_AE and CRF02_AG. For the 17 samples with a viral load below 1000 c/ml and the 104 samples with a viral load above 1000 c/ml, the amplification efficiency was respectively 53% and 94%. The sensitivity of the method was further evaluated using limiting dilution of RNA extracted from a plasma pool containing an equimolar mixture of three HIV-1 subtypes (B, C and CRF02_AG) and diluted before and after cDNA generation. Both RNA and cDNA dilution showed comparable sensitivity and equal accuracy in reflecting the subtype distribution of the plasma pool. One single event of in vitro recombination was detected amongst the 41 sequences obtained after cDNA dilution but no indications for in vitro recombination were found after RNA dilution. In conclusion, a two-amplicon strategy and limiting dilution of viral RNA followed by reverse transcription, nested PCR and Sanger sequencing, allows near full genome sequencing of individual HIV-1 RNA molecules. This method will be a valuable tool in the study of virus evolution and recombination

Longitudinal sequencing of HIV-1 infected patients with low-level viremia for years while on ART shows no indications for genetic evolution of the virus

HIV-infected patients on antiretroviral therapy (ART) may present low-level viremia (LLV) above the detection level of current viral load assays. In many cases LLV is persistent but does not result in overt treatment failure or selection of drug resistant viral variants. To elucidate whether LLV reflects active virus replication, we extensively sequenced pol and env genes of the viral populations present before and during LLV in 18 patients and searched for indications of genetic evolution. Maximum likelihood phylogenetic trees were inspected for temporal structure both visually and by linear regression analysis of root-to-tip and pairwise distances. Viral coreceptor tropism was assessed at different time points before and during LLV. In none of the patients consistent indications for genetic evolution were found over a median period of 4.8 years of LLV. As such these findings could not provide evidence that active virus replication is the main driver of LLV

Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART

Background: Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. Objectives: To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. Study design: A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. Results: Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/10(6) blood cells and showed significant correlation with the pre-ART CD4(+) T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. Conclusions: Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution

Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming

ABSTRACTIn vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5’ end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5’ end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5’ end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1

Markers associated with persisting low-level viraemia under antiretroviral therapy in HIV-1 infection

To identify host and viral characteristics associated with long-term persisting low-level viraemia (PLLV) under antiretroviral therapy (ART). Seventy-one ART-treated patients with long-term PLLV (20250 copies/mL) and 102 control patients with systematically undetectable viral load (VL) were selected retrospectively from ART-treated patients followed at the Ghent HIV reference centre. Host and viral characteristics were compared using univariate and multivariate analyses. Higher plasma VL at therapy initiation (OR 3.52; 95 CI 1.866.65; P0.001), therapy re-initiation after an interruption (OR 3.94; 95 CI 1.709.16; P0.001), male gender (OR 4.28; 95 CI 1.4013.00; P0.011), a protease inhibitor-based regimen (OR 2.90; 95 CI 1.206.97; P0.017) and predicted CCR5 co-receptor tropism (OR 2.53; 95 CI 1.056.11; P0.039) were independently associated with PLLV. VL at ART initiation, therapy history, gender, ART regimen and co-receptor tropism were independently associated with PLLV. Gender, therapy history, co-receptor tropism and VL at ART initiation could be valuable predictive markers to identify patients at risk for PLLV

Drug resistance is rarely the cause or consequence of long-term persistent low-level viraemia in HIV-1-infected patients on ART

Background: The introduction of highly sensitive HIV-1 viral load assays with a lower quantification limit of 20 copies/ml uncovered that in a number of patients on ART, the viral load systematically fluctuates around or slightly above the detection limit of the assays. This study aimed to analyse the presence or occurrence of drug resistance mutations in HIV-1-infected patients during long-term persistent low-level viraemia (PLLV) under ART. Methods: A retrospective study was carried out in which baseline and on-therapy presence of drug resistance mutations in the HIV-1 protease and reverse transcriptase genes were analysed in patients with PLLV between 20 and 250 copies/ml. For all available plasma samples collected during PLLV, resistance analysis was attempted with an ultrasensitive amplification and sequencing protocol. Results: Resistance analysis was successful for 154 samples collected longitudinally from 23 patients over a median period of 4.7 years (IQR 3.3-5.7). Twenty of these patients were on a boosted protease inhibitor (PI)-based regimen (87%). Single drug resistance mutations were detected in isolated samples of 4 patients, 2 of the 3 patients who initiated a non-nucleoside reverse transcriptase inhibitor-based regimen and 2 of the 20 on a PI-based regimen. Only one of the detected mutations decreased susceptibility to the therapy regimen taken at the time of sample collection. Drug resistance mutations were not found in the three patients who developed virological failure (viral load >250 copies/ml) during the study. Conclusions: Long episodes of PLLV in patients on boosted PI-based regimens rarely result in the selection of new drug-resistant variants

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots▿

Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study