Pol Region Research Articles

Correlation of HIV-1 drug resistant mutations and virologic failure

Introductionmutations are important by ensuring that the HIV-1 agent remains fit in the environment and evades drugs that are developed purposely to kill them. In Kenya, mutations conferring resistance to available ARVs have been reported in previous studies. However, there is a paucity of information on whether these previous studies have reported all mutations conclusively that confer resistance to available drugs leading to virologic failure. Therefore, this study was sought to identify the current HIV-1 drug-resistant mutations attributable to virologic failure among adults on various ARV regimens.Methodsthe samples were collected March to June 2020. Analysis of viral loads and HIV-1 drug-resistant mutations through sequencing of the pol region of HIV-1 were done. Alignment of the cDNA sequences was done by Recall (beta version 3.05) software. HIV-1 resistant mutations were identified by Stanford University HIV drug resistance database.Resultsmost of the participants had viral loads of more than 1000 copies/ml during all the three visits. Out of 125 mutations identified, 83 mutations resulted in virologic failure. Out of 17 new mutations, 14 resulted in virologic failure and included NRTIs (L74I, L74V, T69D, V65R); NNRTIs (A98G, V179E, V179F, V179D, 179F); PIs (I54V3, F53L2, L89T, G48A).Conclusionthe study reveals new HIV-1 drug-resistant mutations which have never been reported in Kenya as well as old and both resulted in virologic failure. This calls for frequent monitoring and profiling of mutations that will enable decision-making in the drugs and vaccine design and development.

Оценка эффективности алгоритма выявления случаев недавнего инфицирования ВИЧ-1 в Российской Федерации

Objective. Accurate identification of recent HIV-1 infection cases will ensure a more effective and precise assessment of the dynamics of virus transmission, the time between infection and diagnosis, and the quality of screening and prevention programs. This study was undertaken to adjust the recent HIV-1 infection testing algorithm using a cohort of patients, in whom the time since infection was known. Materials and methods. We used blood plasma samples obtained from 264 HIV-infected patients with a known date of infection. All samples were analyzed using two serological assays aimed to differentiate between cases of recent and established HIV infection. Using the results of sequencing of the pol region, we calculated the proportion of variable positions in order to determine the duration of infection. To identify the cases of recent HIV infection, we evaluated different variants of a diagnostic algorithm that included a combination of serological tests, molecular genetic analysis of the viral genome, and other clinical and laboratory parameters. Results. The effectiveness of the DS-ELISA-HIV-AB-TERM (DS) assay for the detection of recent infection was higher than that of the Architect HIV Ag/Ab Combo assay (Abbott). The sensitivity and specificity of the DS assay were 94.4% and 96.7%, respectively. The sensitivity and specificity of the Abbott assay were 86.4% and 77,4%, respectively. The HIV-1 genome variability threshold of 0.33% allowed the differentiation between samples depending on the time since infection with a cut-off of 12 months: 82.1% of recent samples and 62.7% of established samples were correctly identified using this method. We analyzed the effectiveness of schemes of the algorithm for the detection of recent infection lasting no longer than 9 months. Conclusion. Our findings allow us to recommend the algorithm based on the Russian DS assay for the detection of recent HIV-1 cases in routine clinical practice. This algorithm will enable the detection of new HIV cases, thereby improving the disease control. Key words: HIV-1, HIV infection, genetic variability, time since infection, duration of infection, recent infection, early infection, seroconversion

The genotype distribution, infection stage and drug resistance mutation profile of human immunodeficiency virus-1 among the infected blood donors from five Chinese blood centers, 2014–2017

Human immunodeficiency virus-1 (HIV-1) exhibits high diversity and complexity in China, challenging the disease surveillance and antiretroviral therapy. Between July 1, 2014 and January 30, 2017, we investigated the profiles of HIV-1 infection stages, genotype distribution and drug resistance mutations (DRMs) using plasma samples from HIV Western blot (WB) confirmed blood donors from five Chinese blood centers (Chongqing, Guangxi, Luoyang, Mianyang, and Urumqi). HIV pol regions consisted of whole protease and partial reverse transcriptase were genotyped and analyzed for DRMs. Lag-Avidity testing was performed to identify the infection stages. Of the 356 HIV-1 WB positive samples tested by Lag-avidity assay, 19.1% (68/356) were recent infections. Genotyping on 356 amplified sequences presented the subtype distributions as following: CRF07_BC (65.7%), CRF08_BC (7.3%), CRF01_AE (19.1%), B (4.2%), CRF55_01B (3.1%), CRF59_01B (0.3%) and CRF68_01B (0.3%). No significant difference in genotype distribution was observed between recent and long-term infections. 48 DRMs were identified from 43 samples, indicating a drug resistance prevalence of 12.1% (43/356), which include seven protease inhibitors (PIs) accessory DRMs (Q58E, L23I and I84M), two PIs major DRMs (M46I, M46L), seven nucleoside RT inhibitors DRMs (D67N, K70Q, K219R and M184L), and 32 non-nucleoside RT inhibitors DRMs (K103N, V179E, K238N, V179D, E138G, G190E, A98G, Y188D and E138A). In addition, we had also identified CRFs from the 01B subtype including CRF55_01B (3.1%), CRF59_01B (0.3%) and CRF68_01B (0.3%). As an important part of the continuous monitoring of HIV-1 circulating strains among blood donors, our findings were expected to contribute to the comprehensive AIDS control and development of proper diagnostics for HIV-1 in China.

Non-B variants of HIV-1 in San Francisco, California.

The HIV-1 epidemic in the US has historically been dominated by subtype B. HIV subtype diversity has not been extensively examined in most US cities to determine whether non-B variants have become established, as has been observed in many other global regions. We describe the diversity of non-B variants and present evidence of local transmission of non-B HIV in San Francisco. Viral sequences collected from patients between 2000 and 2016 were matched to the San Francisco HIV/AIDS case registry. HIV subtype was determined using COMET. Phylogenies were reconstructed using the pol region of subtypes A, C, D, G, CRF01_AE, CRF02_AG, and CRF07_BC, with reference sequences from the LANL HIV database. Associations of non-B subtypes and circulating recombinant forms (CRFs) with patient characteristics were assessed using multivariable logistic regression. Out of 11,381 sequences, 10,669 were from 7235 registry cases, of which 141 (2%) had non-B subtypes and CRFs and 72 (1%) had unique recombinant forms. CRF01_AE (0.8%) and subtype C (0.5%) were the most prevalent non-B forms. The frequency of non-B subtypes and CRFs increased in San Francisco during years 2000-2016. Out of 146 transmission events involving non-B study sequences, 18% indicated local transmission within the study population and 74% appeared to be inward migration of the virus. Compared to 7016 cases with only subtype B, 141 cases with non-B sequences were more likely to be of non-US country of birth (aOR = 11.02; p < 0.001), of Asian/Pacific-Islander race/ethnicity (aOR = 3.17; p < 0.001), and diagnosed after 2009 (aOR = 4.81; p < 0.001). Results suggest that most non-B infections were likely acquired outside the US and that local transmission of non-B forms has occurred but so far has not produced extensive transmission networks. Thus, non-B variants were not widely established in San Francisco, an observation that differs from cities worldwide with more diverse epidemics.

Increased HIV-1 pretreatment drug resistance with consistent clade homogeneity among ART-naive HIV-1 infected individuals in Ethiopia

BackgroundThe development of pretreatment drug resistance (PDR) is becoming an obstacle to the success of antiretroviral therapy (ART). Besides, data from developing settings including Ethiopia is still limited. Therefore, this study was aimed to assess HIV-1 genetic diversity and PDR mutations among ART-naive recently diagnosed HIV-1 infected individuals in Addis Ababa, Ethiopia.MethodsInstitutional based cross-sectional study was conducted from June to December 2018 in Addis Ababa among ART-naive recently diagnosed individuals. Partial HIV-1 pol region covering the entire protease (PR) and partial reverse transcriptase (RT) regions of 51 samples were amplified and sequenced using an in-house assay. Drug resistance mutations were examined using calibrated population resistance (CPR) tool version 6.0 from the Stanford HIV drug resistance database and the International Antiviral Society-USA (IAS-USA) 2019 mutation list.ResultsAccording to both algorithms used, 9.8% (5/51) of analyzed samples had at least one PDR Mutation. PDR mutations to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) were the most frequently detected (7.8% and 9.8%, according to the CPR tool and IAS-USA algorithm, respectively). The most frequently observed NNRTIs-associated mutations common to both algorithms were K103N (2%), Y188L (2%), K101E (2%), and V106A (2%), while E138A (2%) was observed according to IAS-USA only. Y115F and M184V (mutations that confer resistance to NRTIs) dual mutations were detected according to both criteria in a single study participant (2%). PDR mutation to protease inhibitors was found to be low (only G73S; 2% according to the CPR tool). Phylogenetic analysis showed that 98% (50/51) of the study participants were infected with HIV-1C virus while one individual (2%) was infected with HIV-1A1 virus.ConclusionsThis study showed an increased level of PDR and persistence HIV-1C clade homogeneity after 15 years of the rollout of ART and 3 decades of HIV-1C circulation in Ethiopia, respectively. Therefore, we recommend routine baseline genotypic drug resistance testing for all newly diagnosed HIV infected patients before initiating treatment. This will aid the selection of appropriate therapy in achieving the 90% of patients having an undetectable viral load in consonance with the UN target.

Yeast DNA polymerase η possesses two PIP-like motifs that bind PCNA and Rad6-Rad18 with different specificities

In translesion synthesis (TLS), specialized DNA polymerases, such as polymerase (pol) η and Rev1, are recruited to stalled replication forks. These polymerases form a multi-protein complex with PCNA, Rad6-Rad18, and other specialized polymerases. Pol η interacts with PCNA and Rev1 via a PCNA-interacting protein (PIP) motif in its C-terminal unstructured region. Here we report the discovery of a second PIP-like motif in the C-terminal region of pol η, which we have designated as PIP2. We have designated the original PIP motif as PIP1. We show that the pol η PIP1 and PIP2 motifs bind PCNA with different affinities and kinetics. PIP1 binds with higher affinity than does PIP2, and PIP1 dissociates more slowly than does PIP2. In addition, we show that the interaction between pol η and Rad6-Rad18 is also mediated by the pol η PIP1 and PIP2 motifs. Again, we show that the affinity and kinetics by which these motifs bind Rad6-Rad18 is different. These findings are significant, because the multiple PIP-like motifs on pol η likely play quite different roles within the multi-protein complex formed at stalled replication forks. PIP1 likely plays a critical role in the recruiting pol η to this multi-protein complex. PIP2, by contrast, likely plays a critical role in maintaining the architecture and the dynamics of this multi-protein complex needed to maximize the efficiency and accuracy of TLS.

Phylogeny of partial gag, pol and env genes show predominance of HIV-1G and CRF02_AG with emerging recombinants in south-eastern Nigeria

Human Immunodeficiency Virus is characterized by high degree of genetic diversity with marked differences in its geographic distribution even within a country. This study was designed to identify the strains of HIV-1 circulating among infected individuals in southeastern parts of Nigeria.Genomic DNA was extracted from blood samples of 30 HIV-1 infected individuals from Anambra, Delta and Imo states of southeastern Nigeria. Portions of the genome corresponding to entire p24 gag, entire protease and C2–V3 env genes were amplified by nested PCR, sequenced using Sanger's method and phylogenetically analysed.Out of the 30 samples sequenced, 17, 28 and 14 readable sequences were obtained for gag, pol and env regions respectively. The most prevalent subtypes were CRF02_AG (41.2% in gag, 57.1% in pol protease and 50.0% in env) and G (29.4% in gag, 35.7% in pol protease and 35.7% in env). Other subtypes identified include A (17.7% in gag, 7.1% in env) and J (7.1% in env). Also 2 sequences each in gag (11.8%) and pol protease (7.1%) regions were unclassified but preliminary analysis showed they are recombinants. Furthermore, 71.4% of the isolates with sequences in the 3 regions and 26.7% of those with sequences in 2 genomic regions were recombinant forms.CRF02_AG and subtype G are the predominant HIV-1 strains circulating among infected individuals in southeastern Nigeria. Preliminary analysis results of unclassified sequences suggest that they are new recombinants.

High nonnucleoside reverse transcriptase inhibitor resistance levels in HIV-1-infected Zambian mother-infant pairs.

To elucidate relationships in antiretroviral resistance between HIV-1-infected mother-infant pairs by defining the resistance profiles in the mothers and infants and quantifying drug resistance prevalence in the pairs post-Option B+ implementation. Collection of dried blood spots from mother-infant pairs during routine HIV-1 screens in Lusaka, Zambia from 2015 to 2018. DNA was extracted from the dried blood spots, the HIV-1 pol region was amplified, and the purified proviral DNA was sequenced using Sanger sequencing. Drug resistance mutations (DRM) were identified in sequenced DNA using the Stanford HIVdb (https://hivdb.stanford.edu/). DRM were detected in 45% (44/97) of samples, and these samples were found to harbor resistance to at least two antiretrovirals. The prevalence of nonnucleoside reverse transcriptase inhibitor resistance was significantly higher than that of other antiretroviral classes. DRM were detected disproportionately in infants (67%; 33/49) compared with mothers (23%; 11/48), but the magnitude of resistance did not differ when resistance was detected. The disparity in drug resistance profiles was reinforced in pairwise comparison of resistance profiles in mother-infant pairs. While Option B+ is effective in reducing mother-to-child transmission, in cases where this regimen fails, high-level nonnucleoside reverse transcriptase inhibitor resistance is frequently detected in infants. This underscores the importance of pretreatment drug resistance screening in both mothers and infants and emphasizes the necessary change to protease inhibitor-based and integrase inhibitor-based regimens for treatment of HIV-1-infected infants and mothers.

First Detection of a Circulating Recombinant Form of HIV-1 CRF01_AE/08_BC (CRF105_0108) with Drug-Resistant Mutations in Sichuan, China.

In this study, we identified a novel circulating recombinant form (CRF) of HIV (CRF105_0108) in a total of five patients with no obvious epidemiological linkage in Sichuan, China. Four strains were found in this study, and another strain (XC2014EU01) was reported in a previous study. The phylogenetic analysis of the five near full-length genomes showed that a CRF08_BC segment was inserted into the CRF01_AE backbone with one recombinant breakpoint in the pol region, which is the first HIV-1 CRF of CRF01_AE and CRF08_BC. Three genetic sequences had drug-resistant mutations, E138Q and V179D, indicating that there were low resistance levels to efavirenz (EFV) and nevirapine (NVP) in Liangshan Yi Autonomous Prefecture. CRF105_0108 increases the diversity and complexity of the prevalent HIV-1 CRFs in Sichuan. Simultaneously, drug-resistant mutations in this CRF may influence the efficacy of highly active antiretroviral treatments (ARTs) in Liangshan Yi Autonomous Prefecture.

Drug level testing as a strategy to determine eligibility for drug resistance testing after failure of ART: a retrospective analysis of South African adult patients on second-line ART.

abstract IntroductionWhen protease inhibitor (PI)‐based second‐line ART fails, guidelines recommend drug resistance testing and individualized third‐line treatment. However, PI‐resistant viral strains are rare and drug resistance testing is costly. We investigated whether less costly PI‐exposure testing can be used to select those patients who would benefit most from drug resistance testing.MethodsWe performed a retrospective analysis of South African adults living with HIV experiencing failure of ritonavir‐boosted‐lopinavir (LPV/r)‐based second‐line ART for whom drug resistance testing results were available. We included patients who received plasma‐based drug resistance testing at a central South African reference laboratory in 2017 and patients who received dried blood spots (DBS)‐based drug resistance testing at a rural South African clinic between 2009 and 2017. PI‐exposure testing was performed on remnant plasma or DBS using liquid chromatography mass spectrometry (LCMS). Additionally, a low‐cost immunoassay was used on plasma. Population genotypic drug resistance testing of the pol region was performed on plasma and DBS using standard clinical protocols.ResultsSamples from 544 patients (494 plasma samples and 50 DBS) were included. Median age was 41.0 years (IQR: 33.3 to 48.5) and 58.6% were women. Median HIV‐RNA load was 4.9 log10 copies/mL (4.3 to 5.4). Prevalence of resistance to the NRTI‐backbone was 70.6% (349/494) in plasma samples and 56.0% (28/50) in DBS. Major PI‐resistance mutations conferring high‐level resistance to LPV/r were observed in 26.7% (132/494) of plasma samples and 12% (6/50) of DBS. PI‐exposure testing revealed undetectable LPV levels in 47.0% (232/494) of plasma samples and in 60.0% (30/50) of DBS. In pooled analysis of plasma and DBS samples, detectable LPV levels had a sensitivity of 90% (84% to 94%) and a negative predictive failure of 95% (91% to 97%) for the presence of major LPV/r resistance.ConclusionsPI‐exposure testing revealed non‐adherence in half of patients experiencing failure on second‐line ART and accurately predicted the presence or absence of clinically relevant PI resistance. PI‐exposure testing constitutes a novel screening strategy in patients with virological failure of ART that can differentiate between different underlying causes of therapy failure and may allow for more effective use of limited resources available for drug resistance testing.

Dendritic cell targeting anti-CD40 antibody-CD40L-HIV-1 vaccines are adjuvant intrinsic

Abstract Soluble CD40L and agonistic anti-CD40 monoclonal antibody are adjuvants used in vaccination settings. Vaccines based on anti-CD40 antibodies fused to HIV-1 antigens are in clinical development. Studies with current anti-CD40-based dendritic cell (DC) targeting vaccines show that co-administration of an adjuvant is needed for maximal immune responses. We show that by fusing CD40L to CD40-targeting antibodies, activation of DCs concomitant with antigen uptake and processing is maximized, and this provides a context CD40-targeting vaccines with intrinsic adjuvant activity. Direct fusion of CD40L to L or H chain C-termini results in CD40 agonists with ‘superagonist’ properties. Especially on DCs, both potency and efficacy for induction of cytokine secretion and activation markers is greatly enhanced compared to known strong agonists like Pfizer’s anti-CD40 CP-870-89 antibody. This potency was maintained by anti-CD40-CD40L constructs fused to HIV-1 antigens from Gag, Nef, and Pol regions (HIV5pep). Anti-CD40-CD40L-HIV5pep preferentially expanded CD8+ T cells from HIV-1+ donor PBMCs compared to the same antibody-antigen fusions without attached CD40L. Anti-CD40-CD40L-TEα and anti-CD40-TEα both evoked robust proliferation of TEα-specific CD4+ T cells in human CD40 transgenic mice, but only anti-CD40-CD40L-TEα vaccine elicited TEα-specific CD4+ T cells producing IFNγ. Also, anti-CD40-CD40L-Env gp140 vaccine without adjuvant in human CD40 transgenic mice elicited stronger anti-Env gp140 antibody responses than anti-CD40-Env gp140 vaccine. Thus superagonist anti-CD40 antibodies directly fused to the natural ligand show great advantage in inducing immune responses without the use of an extrinsic adjuvant.

Near Full-Length Genomic Characterization of a Novel Unique Recombinant (CRF01_AE/CRF07_BC) in Fuyang City of China.

Recombinant forms contribute substantially to the genetic diversity of HIV-1. Recent studies have also revealed that three major viral strains (CRF07_BC, CRF01_AE, and subtype B) have been cocirculating among men who have sex with men (MSM) in Anhui, suggesting a high probability of generating new recombinants. In this study, we reported a novel CRF01_AE and CRF07_BC HIV-1 recombinant form in MSM in Fuyang city of China. Two near full-length genome (NFLG) named FY184 and FY208 were successfully obtained. The genomic composition analysis of the NFLG reveals that it was divided into four segments by three breakpoints, with two regions of CRF07_BC inserted into a CRF01_AE backbone's gag and pol regions. The CRF01_AE regions were originated from a subcluster lineage of CRF01_AE, which is mainly circulating among MSM in China. The emergence of a novel recombinant of CRF01_AE/CRF07_BC is indicative of the increasing genetic diversity of the HIV epidemic in MSM in Anhui.

Phylogenetic Analysis of Belgian Small Ruminant Lentiviruses Supports Cross Species Virus Transmission and Identifies New Subtype B5 Strains.

Small ruminant lentiviruses (SRLV) are a group of highly divergent viruses responsible for global and fatal infections in sheep and goats. Since the current phylogenetic classification of these viruses was proposed in 2004, it nowadays consists out of 5 genotypes and 28 subtypes. In support of our national SRLV control program, we performed the genetic characterization of SRLV strains circulating in the Belgian sheep and goat population. Fourteen sheep and 9 goat strains were sequenced in the gag-pol and pol regions using the method described by Shah. Most SRLV strains from sheep and goats belonged to prototype A1 and B1 subtypes, respectively. We, however, also found indications for cross-species transmission of SRLV strains between sheep and goats and vice versa, and identified a new subtype designated as B5. An in-depth analysis of the current SRLV phylogeny revealed that many subtypes have been defined over the years based on limited sequence information. To keep phylogeny as a useful tool, we advocate to apply more rigorous sequencing standards to ensure the correct classification of current and new emerging strains. The genetic characterization of Belgian SRLV strains will help in the development of appropriate diagnostic tools to assist the national control program.

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

Comprehensive PCR assays for the genotypic drug resistance analysis of all HIV-1 antiretroviral agents (reverse transcriptase, protease and integrase inhibitors) are increasingly in demand due to introduction of integrase inhibitors in the first line regimens and the increasing presence of non-B HIV-1 clades around the world. This study focused on the development and evaluation of a new PCR-based assay for the amplification and sequencing of the entire HIV-1 pol region of major circulating group M HIV-1 strains in Europe for genotypic drug resistance analysis. The comprehensive touchdown PCR assay developed in this study utilized HIV-1 RNA extracted from the plasma of blood samples of consenting HIV-1 infected patients in Cyprus, collected from 2017 to 2019. The HIV-1 pol region was amplified by touchdown PCR for both the primary RT-PCR and the secondary PCR steps. Successful PCR amplicons were determined by population DNA sequencing, using the Sanger method and the genotypic drug resistance analysis was performed with the Stanford University HIV Drug Resistance Database Program. The newly developed assay successfully amplified the entire HIV-1 pol region (2844 nucleotides long) of 141 out of 144 samples of group M HIV-1 subtypes and recombinant strains of the Cyprus HIV-1 Transmission Cohort Study (CHICS) isolated from 2017 to 2019 and genotypic analyses were conducted for all currently available HIV-1 reverse transcriptase, protease and integrase inhibitors. The drug resistance, epidemiological and demographic data of these study subjects will be expanded upon in the CHICS (L.G. Kostrikis et al., manuscript in preparation for publication). The newly developed HIV-1 genotypic drug resistance assay would benefit clinical settings, and research focusing on the world-wide spread of HIV-1 drug-resistant strains, especially in geographic regions characterized by polyphyletic HIV-1 infections.

Evaluation of Phylogenetic Methods for Inferring the Direction of Human Immunodeficiency Virus (HIV) Transmission: HIV Prevention Trials Network (HPTN) 052.

Phylogenetic analysis can be used to assess human immunodeficiency virus (HIV) transmission in populations. We inferred the direction of HIV transmission using whole-genome HIV sequences from couples with known linked infection and known transmission direction. Complete next-generation sequencing (NGS) data were obtained for 105 unique index-partner sample pairs from 32 couples enrolled in the HIV Prevention Trials Network (HPTN) 052 study (up to 2 samples/person). Index samples were obtained up to 5.5 years before partner infection; partner samples were obtained near the time of seroconversion. The bioinformatics method, phyloscanner, was used to infer transmission direction. Analyses were performed using samples from individual sample pairs, samples from all couples (1 sample/person; group analysis), and all available samples (multisample group analysis). Analysis was also performed using NGS data from defined regions of the HIV genome (gag, pol, env). Using whole-genome NGS data, transmission direction was inferred correctly (index to partner) for 98 of 105 (93.3%) of the individual sample pairs, 99 of 105 (94.3%) sample pairs using group analysis, and 31 of the 32 couples (96.9%) using multisample group analysis. There were no cases where the incorrect transmission direction (partner to index) was inferred. The accuracy of the method was higher with greater time between index and partner sample collection. Pol region sequences performed better than env or gag sequences for inferring transmission direction. We demonstrate the potential of a phylogenetic method to infer the direction of HIV transmission between 2 individuals using whole-genome and pol NGS data.

The dual-target approach in viral HIV-1 viremia testing: An added value to virological monitoring?

New methods of HIV-1 RNA quantification based on dual-target detection are increasingly used in HIV viral load monitoring, but clinical implications and impact of dual-target detection on HIV-1 infection management are not established. Aptima HIV-1 Quant Dx assay is a last generation HIV viral load method, that uses pol and LTR as simultaneous target, providing quantitative results based mainly on pol target, while LTR target is used to report the results when pol signal is absent. In our laboratory, about 6% of results of all HIV-1 viral load tests performed with this platform in one year period resulted from LTR signal. Interestingly, LTR-based viremia (sometimes exceeding 1,000 copies/mL) was observed in a small proportion (up to 1%) of patients under ART, considered for long time virologically suppressed on the basis of a single target (pol-based) assay. Male gender, >700 vs <200 CD4 cell/mL and dual therapy including NRTI plus either NNRTI, or PI/b or INSTI were independently associated with increased risk of LTR-based HIV-1 viral load detection by multivariable logistic regression. A significant linear correlation was observed between LTR-based HIV-1 RNA levels and PBMC-associated proviral DNA. Moreover, in a small group of patients with HIV-1 RNA levels >200 copies/mL, longitudinal assessments showed parallel kinetics between plasma viremia and proviral DNA. Sequencing of pol region for drug resistance assessment in patients with LTR-based viremia failed on plasma HIV-1 RNA, while it was successful on proviral DNA. The detection/quantification of HIV-1 viremia based only on LTR signal with a dual target assay in samples resulting undetectable with the more conventional target pol needs accurate evaluation; unravelling the biological basis of this phenomenon, here described for the first time, is mandatory to establish relevance and implication by both pathogenetic (i.e. infectivity of LTR-detected viruses, reservoir turnover, immune activation, etc.) and clinical standpoint.

Revealing an Internal Stabilization Deficiency in the DNA Polymerase β K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography

The human DNA polymerase (pol) β cancer variant K289M has altered polymerase activity in vitro, and the structure of wild-type pol β reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF2 group substitution for the β-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol β is not altered in the presence of the dGTP CF2 analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol β and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.

Comparison of Rapid Diagnosis Test and Nested PCR for HIV Diagnosis in Kinshasa

Background: The need for appropriate methods for the diagnosis, monitoring and treatment of HIV infection is increasing in resource-poor settings. Objective: The main objective of this study was to compare the quality of the results of HIV diagnosis by Classical Nested PCR and Rapid Screening Tests (RDTs) in order to contribute to the improvement of the care of People Living with HIV (PLHIV). Methods: The present study is a cross-sectional study that was conducted in Kinshasa. Only people willing to voluntary testing for HIV were selected for this study. Our sample consisted of 100 individuals for voluntary testing for HIV, and of 50 PLHIV who came for their medical appointment. A minimum of 5.0 ml of blood was collected into tubes containing EDTA. After the RDT, the collected blood was transferred at the Laboratory for molecular analysis. The revelation of the amplification results was made under UV light after electrophoretic migration on agarose gel. Results: On D0, 60 samples were positive and 40 negative for HIV by RDT. After amplification by Nested DNA PCR, on D0, the gag region gave 65 positive and 35 negative; while the amplification on the pol region gave 63 positive and 37 negative. At the M6, for patients under treatment, 10 samples were positive and 40 negative for HIV using the RDT. After amplification, all the 50 samples collected were positive by Nested DNA PCR on the gag and pol regions. Conclusion: Nested DNA PCR is precise, specific and accurate that RDT especially for the cases of patients under treatment.

Pervasive and non-random recombination in near full-length HIV genomes from Uganda.

Recombination is an important feature of HIV evolution, occurring both within and between the major branches of diversity (subtypes). The Ugandan epidemic is primarily composed of two subtypes, A1 and D, that have been co-circulating for 50 years, frequently recombining in dually infected patients. Here, we investigate the frequency of recombinants in this population and the location of breakpoints along the genome. As part of the PANGEA-HIV consortium, 1,472 consensus genome sequences over 5 kb have been obtained from 1,857 samples collected by the MRC/UVRI & LSHTM Research unit in Uganda, 465 (31.6 per cent) of which were near full-length sequences (>8 kb). Using the subtyping tool SCUEAL, we find that of the near full-length dataset, 233 (50.1 per cent) genomes contained only one subtype, 30.8 per cent A1 (n = 143), 17.6 per cent D (n = 82), and 1.7 per cent C (n = 8), while 49.9 per cent (n = 232) contained more than one subtype (including A1/D (n = 164), A1/C (n = 13), C/D (n = 9); A1/C/D (n = 13), and 33 complex types). K-means clustering of the recombinant A1/D genomes revealed a section of envelope (C2gp120-TMgp41) is often inherited intact, whilst a generalized linear model was used to demonstrate significantly fewer breakpoints in the gag–pol and envelope C2-TM regions compared with accessory gene regions. Despite similar recombination patterns in many recombinants, no clearly supported circulating recombinant form (CRF) was found, there was limited evidence of the transmission of breakpoints, and the vast majority (153/164; 93 per cent) of the A1/D recombinants appear to be unique recombinant forms. Thus, recombination is pervasive with clear biases in breakpoint location, but CRFs are not a significant feature, characteristic of a complex, and diverse epidemic.

Phylogenetic Analysis of Human Immunodeficiency Virus from People Who Inject Drugs in Indonesia, Ukraine, and Vietnam: HPTN 074.

HIV Prevention Trials Network (HPTN) 074 evaluated human immunodeficiency virus (HIV) prevention interventions for people who inject drugs (PWID) in Indonesia, Ukraine, and Vietnam. Study interventions included support for HIV infection and substance use treatment. The study enrolled index participants living with HIV and injection partners who were not living with HIV. Seven partners acquired HIV infection during the study (seroconverters). We analyzed the phylogenetic relatedness between HIV strains in the cohort and the multiplicity of infection in seroconverters. Pol region consensus sequences were used for phylogenetic analysis. Data from next-generation sequencing (NGS, env region) were used to evaluate genetic linkage of HIV from the 7 seroconverters and the corresponding index participants (index-partner pairs), to analyze HIV from index participants in pol sequence clusters, and to analyze multiplicity of HIV infection. Phylogenetic analysis of pol sequences from 445 index participants and 7 seroconverters identified 18 sequence clusters (2 index-partner pairs, 1 partner-partner pair, and 15 index-only groups with 2-7 indexes/cluster). Analysis of NGS data confirmed linkage for the 2 index-partner pairs, the partner-partner pair, and 11 of the 15 index-index clusters. The remaining 5 seroconverters had infections that were not linked to the corresponding enrolled index participant. Three (42.9%) of the 7 seroconverters were infected with more than 1 HIV strain (3-8 strains per person). We identified complex patterns of HIV clustering and linkage among PWID in 3 communities. This should be considered when designing strategies for HIV prevention for PWID. NCT02935296.