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

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

Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections

<p>Abstract</p> <p>Background</p> <p>The number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission.</p> <p>Methods</p> <p>Baseline HIV-1 <it>pol </it>sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics.</p> <p>Results</p> <p>Multivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with <it>Chlamydia </it>(p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), <it>Chlamydia </it>infection (p = 0.013) and primary HIV (p = 0.017).</p> <p>Conclusions</p> <p>Combination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.</p

Deep sequencing of HIV-1 RNA and DNA in newly diagnosed patients with baseline drug resistance showed no indications for hidden resistance and is biased by strong interference of hypermutation

Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant

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

Frequency of occurrence of HIV-1 dual infection in a Belgian MSM population

<div><p>Introduction</p><p>HIV-1 dual infection is a condition that results from infection with at least two HIV-1 variants from different sources. The scarceness of information on this condition is partly due to the fact that its detection is technically challenging. Using next-generation sequencing we defined the extent of HIV-1 dual infection in a cohort of men who have sex with men (MSM).</p><p>Material & methods</p><p>Eighty-six MSM, diagnosed with HIV-1 subtype B infection between 2008 and 2013 were selected for next-generation sequencing of the HIV-1 <i>envelope</i> V3. Sequencing was performed on 2 plasma samples collected with an interval of > 6 months before the initiation of antiretroviral therapy. Maximum likelihood phylogenetic trees were inspected for dual infection, defined as the presence of two or more monophyletic clusters with ≥ 90% bootstrap support and a mean between-cluster genetic distance of ≥ 10%. To confirm dual infection, deep V3 sequencing of intermediate samples was performed as well as clonal sequencing of the HIV-1 <i>protease-reverse transcriptase</i> gene.</p><p>Results</p><p>Five of the 74 patients (6.8%) for whom deep sequencing was successful, showed clear evidence of dual infection. In 4 of them, the second strain was absent in the first sample but occurred in subsequent samples. This was highly suggestive for superinfection. In 3 patients both virus variants were of subtype B, in 2 patients at least one of the variants was a subtype B/non-B recombinant virus.</p><p>Conclusions</p><p>Dual infection was confirmed in 6.8% of MSM diagnosed with HIV-1 in Belgium. This prevalence is probably an underestimation, because stringent criteria were used to classify viral variants as originating from a new infection event.</p></div

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

BACKGROUND: Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. METHODS: The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. RESULTS: Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. CONCLUSIONS: Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia

Individual phylogenetic trees of the 4 patients with dual infection from whom more than 2 longitudinal samples were analyzed.

<p><b>(A)</b> Patient 66, <b>(B)</b> Patient 35, <b>(C)</b> Patient 16, <b>(D)</b> Patient 08. Bootstrap values ≥ 90% are displayed. Black diamond = HXB2.</p

Characteristics of the 5 patients with HIV-1 dual infection.

<p>Characteristics of the 5 patients with HIV-1 dual infection.</p