HIV-1 Quasispecies Research Articles

Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations-Application to HIV-1 quasispecies.

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing, which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence polymerase-chain reaction (PCR) amplicons derived from cDNA templates tagged with unique molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR. The use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Production of highly accurate sequences from the large datasets produced from SMRT-UMI sequencing is facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline). PORPIDpipeline automatically filters and parses circular consensus reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination, heteroduplex formation, or early cycle PCR errors. The optimized SMRT-UMI sequencing and PORPIDpipeline methods presented here represent a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus quasispecies in a virus transmitter-recipient pair of individuals.

ClusterV-Web: a user-friendly tool for profiling HIV quasispecies and generating drug resistance reports from nanopore long-read data.

Third-generation long-read sequencing is an increasingly utilized technique for profiling human immunodeficiency virus (HIV) quasispecies and detecting drug resistance mutations due to its ability to cover the entire viral genome in individual reads. Recently, the ClusterV tool has demonstrated accurate detection of HIV quasispecies from Nanopore long-read sequencing data. However, the need for scripting skills and a computational environment may act as a barrier for many potential users. To address this issue, we have introduced ClusterV-Web, a user-friendly web-based application that enables easy configuration and execution of ClusterV, both remotely and locally. Our tool provides interactive tables and data visualizations to aid in the interpretation of results. This development is expected to democratize access to long-read sequencing data analysis, enabling a wider range of researchers and clinicians to efficiently profile HIV quasispecies and detect drug resistance mutations. ClusterV-Web is freely available and open source, with detailed documentation accessible at http://www.bio8.cs.hku.hk/ClusterVW/. The standalone Docker image and source code are also available at https://github.com/HKU-BAL/ClusterV-Web.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/μL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

PP 1.43 – 00206 Evaluating integrated HIV-1 quasispecies using Near Full Length sequencing in ART-suppressed individuals in the Drexel CARES Cohort

PP 1.43 – 00206 Evaluating integrated HIV-1 quasispecies using Near Full Length sequencing in ART-suppressed individuals in the Drexel CARES Cohort

Analysis of HIV quasispecies and virological outcome of an HIV D+/R+ kidney\u2013liver transplantation

IntroductionTransplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney–liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring.MethodsThe donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts.ResultsDonor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up.ConclusionsNo evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.

In chronic infection, HIV gag-specific CD4+ T cell receptor diversity is higher than CD8+ T cell receptor diversity and is associated with less HIV quasispecies diversity.

Cellular immune responses to Gag correlate with improved HIV viral control. The full extent of cellular immune responses comprise both the number of epitopes recognized by CD4+ and CD8+ T cells, as well as the diversity of the T cell receptor (TCR) repertoire directed against each epitope. The optimal diversity of the responsive TCR repertoire is unclear. Therefore, we evaluated the TCR diversity of CD4+ and CD8+ T cells responding to HIV-1 Gag to determine if TCR diversity correlates with clinical or virologic metrics. Previous studies of TCR repertoires have been limited primarily to CD8+ T cell responses directed against a small number of well-characterized T cell epitopes restricted by specific human leucocyte antigens. We stimulated peripheral blood mononuclear cells from 21chronic HIV-infected individuals overnight with a pool of HIV-1 Gag peptides, followed by sorting of activated CD4+ and CD8+ T cells and TCR deep sequencing. We found Gag-reactive CD8+ T cells to be more oligoclonal, with a few dominant TCRs comprising the bulk of the repertoire, compared to the highly diverse TCR repertoires of Gag-reactive CD4+ T cells. HIV viral sequencing of the same donors revealed that high CD4+ T cell TCR diversity was strongly associated with lower HIV Gag genetic diversity. We conclude that the TCR repertoire of Gag-reactive CD4+ T helper cells display substantial diversity without a clearly dominant circulating TCR clonotype, in contrast to a hierarchy of dominant TCR clonotypes in the Gag-reactive CD8+ T cells, and may serve to limit HIV diversity during chronic infection.IMPORTANCE Human T cells recognize portions of viral proteins bound to host molecules (human leucocyte antigens) on the surface of infected cells. T cells recognize these foreign proteins through their T cell receptors (TCRs), which are formed by the assortment of several available V, D and J genes to create millions of combinations of unique TCRs. We measured the diversity of T cells responding to the HIV Gag protein. We found the CD8+ T cell response is primarily made up of a few dominant unique TCRs whereas the CD4+ T cell subset has a much more diverse repertoire of TCRs. We also found there was less change in the virus sequences in subjects with more diverse TCR repertoires. HIV has a high mutation rate, which allows it to evade the immune response. Our findings describe the characteristics of a virus-specific T cell response that may allow it to limit viral evolution.

Early-Transmitted Variants and Their Evolution in a HIV-1 Positive Couple: NGS and Phylogenetic Analyses.

We had access to both components of a couple who became infected with human immunodeficiency virus (HIV)-1 through sexual behavior during the early initial phase of infection and before initiation of therapy. We analyzed blood samples obtained at the time of diagnosis and after six months of combined antiretroviral therapy. Next-generation sequencing (NGS) and phylogenetic analyses were used to investigate the transmission and evolution of HIV-1 quasispecies. Phylogenetic analyses were conducted using Bayesian inference methods. Both partners were infected with an HIV-1 B subtype. No evidence of viral recombination was observed. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (distances ranging from 0.102 to 0.148). One HIV-1 single variant was concluded to be dominant in all of the HIV-1 regions analyzed, although some minor variants could be observed. The same tree structure was observed both at baseline and after six months of therapy. These are the first extended phylogenetic analyses performed on both members of a therapy-naïve couple within a few weeks of infection, and in which the effect of antiretroviral therapy on viral evolution was analyzed. Understanding which HIV-1 variants are most likely to be transmitted would allow a better understanding of viral evolution, possibly playing a role in vaccine design and prevention strategies.

Increased acquired protease inhibitor drug resistance mutations in minor HIV-1 quasispecies from infected patients suspected of failing on national second-line therapy in South Africa

BackgroundHIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs.MethodsDuring 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient’s treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database.ResultsStatistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p = 0.042) and integrase inhibitor RAM (4%; 2/56; p = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation (n = 13) in protease and K65R (n = 5), K103N (n = 7) and M184V (n = 5) in reverse transcriptase.ConclusionsHTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in < 20% of the population. Deep sequencing could be of greater value in detecting acquired resistance mutations early.

Dynamics of HIV-1 quasispecies diversity of participants on long-term antiretroviral therapy based on intrahost single-nucleotide variations

ObjectivesHuman immunodeficiency virus (HIV) quasispecies diversity presents a large barrier to the eradication of HIV. The aim of this study was to investigate intrahost HIV quasispecies diversity and evolutionary patterns underpinning the mechanisms of viral pathogenesis during antiretroviral therapy (ART). MethodsForty-five participants with HIV-1 infection were enrolled in a follow-up cohort for >84 months in 2004, and received a lamivudine-based first-line ART regimen. Blood samples were collected every 6 months to measure viral load and CD4+ cell count. Ultra-deep sequencing and phylogenetic analysis were used to characterize the dynamics governing quasispecies diversity of HIV-1 circulating between plasma RNA and cellular DNA of participants with treatment failure (TF, n = 20) or virologic suppression (VS, n = 25). ResultsAnalysis of the distribution of intrahost single-nucleotide variations (iSNVs) and their mutated allele frequencies revealed that approximately 65% of the quasispecies co-occurred in plasma HIV RNA and cellular DNA either before or after ART. The number and frequency of iSNVs are more representative of intrahost HIV diversity, and have better generalizability than phylogenetic inference by measurement of phylogenetic associations. Furthermore, drug-resistance-associated mutations (DRAMs) accumulated to high levels, dramatically increasing the DRAM-to-total-mutation ratio for TF patients. Linear regression analysis revealed that emergent mutations accumulated faster in TF patients compared with VS patients, at a rate of 0.02 mutations/day/kb. ConclusionsBased on iSNV analysis, the results demonstrate the dynamics of intrahost HIV quasispecies diversity in patients on ART, and provide a novel insight into the persistence of HIV and development of DRAMs.

The inference of HIV-1 transmission direction between a man who has sex with men and his heterosexual wife based on the sequences of HIV-1 quasi-species

ABSTRACT Currently, homosexual transmission has become one of the main routes of HIV-1 spread in China. Furthermore, about 80% Chinese men, who have sex with men (MSM), feel forced to enter eventually into heterosexual marriages due to the Chinese traditional marriage culture, which may cause HIV-1 infection in families. In this study, we identified HIV-1 transmission in a family and the direction of HIV-1 transmission from a MSM to his wife and infant, which indicated Chinese MSM may have become a potential bridge of HIV-1 transmission to their wives and children. Therefore, we need to develop more effective defence measures to prevent the spread of HIV-1 in MSM families in China.

Resistance profile of HIV-1 quasispecies in patients under treatment failure using single molecule, real-time sequencing.

Short-read next-generation sequencing (NGS) has been implemented to study the resistance profile of HIV as it provides a higher sensitivity than Sanger sequencing. However, short-reads only generates a consensus view of the viral population rather than a reconstruction of the viral haplotypes. In this study, we evaluated the resistance profile of HIV quasispecies in patients undergoing treatment failure using SMRT sequencing. Whole-pol RT-PCR was performed on viral RNA extracted from plasma samples of 38 HIV-positive individuals undergoing treatment failure, and sequenced in the RSII instrument. Error correction and viral haplotype phasing was performed with the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm. Presence of resistance mutations reported by the IAS-USA in 2017 was assessed using an in-house script. The SMRT sequencing-based test detected 131/134 resistance mutations previously detected using a Sanger sequencing-based test. However, the SMRT test also identified seven additional mutations present at an estimated frequency lower than 30%. The intra-host phylogenetic analysis showed that seven samples harbored at least one resistance variant at 20--80% frequency. The haplotype-resolved sequencing revealed viral diversification and selection of new resistance during suboptimal treatment, an overall trend toward selection and accumulation of new resistance mutations, as well as the co-existence of resistant and susceptible variants. Our results validate the SMRT sequencing-based test for detection of HIV drug resistance. In addition, this method unraveled the complex dynamic of HIV quasispecies during treatment failure, which might have several implications on clinical management.

Near point-of-care, point-mutation test to detect drug resistance in HIV-1: a validation study in a Mexican cohort.

Pretreatment HIV-drug resistance (PDR, HIVDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is increasing globally. NNRTIs continue to be used as first-line antiretroviral therapy (ART) in some communities due to the cost of dolutegravir-based ART or dolutegravir-associated adverse events. A simplified version of the oligonucleotide ligation assay (OLA) - 'OLA-Simple' - is a low-cost, near point-of-care assay that provides ready-to-use lyophilized reagents and reports HIVDR mutations as colored lines on lateral flow strips. Our objective was to design and validate OLA-Simple for a Mexican cohort. OLA-Simple probes to detect K65R, K103N/S, Y181C, M184V, and G190A were optimized for HIV Mexican sequences. Sixty clinical plasma specimens were analyzed by OLA-Simple by technicians blinded to Illumina-MiSeq sequences, and HIVDR results were compared. Plasma RNA was tested using OLA-Simple kits. OLA-Simple lateral flow strips were read by in-house software, and were classified as mutant or wild-type at each codon. The comparison of results by OLA-Simple and Miseq was used to generate receiver-operating characteristic curves. OLA-Simple PCR amplified 59 of 60 specimens and successfully genotyped 287 of 295 codons, with eight of 295 (2.7%) indeterminate results. Compared to MiSeq, OLA-Simple gave five of 295 (1.7%) false-positive and four of 295 (1.4%) false-negative results. Excluding indeterminate results, OLA-Simple classified mutant with an accuracy of 97.4 and 98.8% when using thresholds at 10 and 25% mutant within an individual's HIV quasispecies, respectively. Compared to MiSeq, OLA-Simple detected HIVDR with high sensitivity and accuracy. OLA-Simple could expand access to affordable and rapid HIVDR testing to guide appropriate ART choices in populations using NNRTI-based ART.

The Genetic Diversity of HIV-1 Quasispecies Within Primary Infected Individuals.

HIV has remarkable genetic diversity among populations. The diversity has critical impacts on transmission, immune escape, pathogenesis, and clinical management. HIV-1 diversity originates from frequent mutation and recombination during reverse transcription. This work focuses on the quasispecies genetic dynamics within individuals with primary infections. Eleven men who have sex with men from the Beijing PRIMO Clinical Cohort were identified as primary infection and had three or four series of their anticoagulant blood samples collected. Viral RNA was extracted and amplified using single-genome amplification. Products of the gp120 gene that met single-genome amplification requirements were sequenced. Subtype assortment of all collected sequences was performed using both the jumping profile hidden Markov model (jpHMM) and REGA. Quasispecies diversity at each time was estimated using Mega 6. Intrapatient recombination was analyzed using RDP4. According to the Fiebig classification system, YA-81 belongs to stage III and YA-113 belongs to stage IV. The other samples are all associated with the infection stage of V/VI. YA113 had a dual infection with subtype B and a new unique recombinant form involving CRF01_AE and C. The other eight were infected with CRF01_AE, one was infected with B/C recombinant, and the last one with B. Of the 10 single infections, 8 were caused by 1 founder virus. They all displayed a sharp increase of quasispecies diversity during the sampling times. Two were caused by at least two founder viruses. The diversity of these strains starts at a significantly high level and is followed by a relatively steady trend. Critically, the separate subtypes YA113-B and YA113-CRF01_AE/C both showed a similar trend to those infected by a single founder virus. Recombination analysis revealed that 5 of 11 cases underwent detectable intrapatient recombination. These findings indicate that tracing the dynamics of HIV-1 quasispecies during early infection may be relevant and valuable for understanding pathways of viral diversification and immune escape.

Pre-treatment HIV-drug resistance associated with virologic outcome of first-line NNRTI-antiretroviral therapy: A cohort study in Kenya

BackgroundPre-treatment HIV-drug-resistance (PDR) to WHO-recommended 1st-line non-nucleoside reverse transcriptase inhibitors (NNRTI)-based antiretroviral treatment (ART) is increasing in low-resource communities. We evaluated the risk of PDR on treatment failure if detected at single or multiple codons, at minority (2–9%) or higher (≥10%) frequencies during efavirenz- vs. nevirapine-ART. MethodsWe conducted a pooled analysis across three cohorts of Kenyans initiating 1st-line NNRTI-ART between 2006 and 2014. Mutations K103N, Y181C, G190A, M184V and K65R were detected by an oligonucleotide ligation assay (OLA) and confirmed by Sanger and next-generation sequencing (NGS). PDR was defined as detection of any mutation by OLA when confirmed by NGS. Treatment failure, defined as plasma HIV RNA ≥400 copies/mL at month-12 of ART, was compared by PDR genotypes. FindingsPDR was detected in 59/1231 (4·8%) participants. Compared to wild-type genotypes, PDR in participants prescribed nevirapine-ART was associated with increased treatment failure [PDR 69·2% (27/39) vs. wild-type 10·4% (70/674); p = 0·0001], whether detected as minority [66·7% (4/6)] or higher [69·7% (23/33)] frequencies in an individual's HIV quasispecies (p = 0·002 and p < 0·0001, respectively), or mutations at single [50·0% (12/24)] or multiple [100·0% (15/15)] codons (p < 0·0001). During efavirenz-ART, PDR was also associated with increased virologic failure [PDR 25·0% (5/20) vs. wild-type 5·0% (25/498); p = 0·005], but only if detected at multiple drug-resistant codons [50·0% (3/6); p = 0·003] or high frequencies PDR [33·3% (5/15); p = 0·001]. InterpretationThe risk that PDR confers for treatment failure varies by number of mutant codons and their frequency in the quasispecies, with a lower risk for efavirenz- compared to nevirapine-based regimens. PDR detection and management could extend the effective use of efavirenz-ART in low-resource settings. FundingNIH, PEPFAR.

PP 3.3 - The dynamics of HIV-1 quasispecies diversity circulating in the plasma RNA and cellular DNA of patients with ART

PP 3.3 - The dynamics of HIV-1 quasispecies diversity circulating in the plasma RNA and cellular DNA of patients with ART

Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients

The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.

Intra-host dynamics and co-receptor usage of HIV-1 quasi-species in vertically infected patients with phenotypic switch

HIV-1 infection through vertical transmission provides a good model to evaluate intra-host viral evolution and allows to gain insight into the dynamics of viral populations. Our aim was to assess the diversity and dynamics of X4- and R5-using HIV-1 variants in vertically infected children who presented a switch in SI/ NSI phenotype in MT-2 cell assays during chronic infection. Through molecular cloning and next generation sequencing of the C2-V5 env fragment, we investigated HIV-1 evolution and co-receptor usage based on V3 loop prediction bioinformatic tools of longitudinal samples obtained from 4 children. In all cases, the phylogenetic relationships were assessed by Maximum-Likelihood trees constructed with MEGA 6.0. In two cases, V3 loop sequences predicted exclusively R5-using and or X4-using strains, while in another two a higher degree of concordance was observed between the phenotypic and genotypic characteristics. In 3 of the 4 cases, C2-V5 env sequences from different time points were intermingled in phylogenetic trees, with no segregation neither by time or tropism. In only one case monophyletic clustering defined groups of sequences with different co-receptor usage. Comparison of amino acid frequency between isolates with SI and NSI phenotype allowed the identification of 9 possible genetic determinants in subtype F C2-V5 region of env associated to SI/ NSI phenotype in these patients, one of which had previously been reported for subtype B. Overall, we found a low degree of correlation between phenotypic and genotypic properties of HIV-1 quasispecies in patients under chronic infection. Whether HIV-1 subtype or other factors influence the evolution of HIV-1 in vivo will require further research.

The inference of HIV-1 transmission direction between HIV-1 positive couples based on the sequences of HIV-1 quasi-species

BackgroundTo infer transmission direction of a HIV transmission chain is helpful not only in legal jurisdiction but also in precise intervention to prevent HIV spread. Recently, the direction of transmission is inferred by whether paraphyletic-monophyletic (PM) or a combination of paraphyletic and polyphyletic (PP) topologies is observed or not between the sequences of source and recipient in the phylogenetic tree. However, paraphyly between them often declines over time and may disappear between spouses due to bidirectional transmission after primary infection. In this study, our aim is to test the reliability of inferring HIV transmission direction between epidemiologically linked HIV-1 positive couples using whether or not paraphyly is observed in phylogenetic tree.MethodsHIV quasi-species were sequenced using PCR product clones, and then Bayesian analysis of molecular sequences with MCMC was employed to construct phylogenetic relationship of env, gag, pol gene fragments of HIV-1 positive couples using BEAST software.ResultsOur results showed that all sequences of seven couples except pol sequences of couple 12 and 13 form their own monophyletic cluster in phylogenetic tree including the closest control sequences from GenBank or other studies on local samples, which are supported by significant Bayesian posterior probabilities more than 0.9932. Of seven couples, paraphyly is only observed in phylogenetic tree constructed with env and pol gene sequences of three couples and gag gene sequences of four couples. Paraphyly is not observed in half of HIV positive couples. Pol sequences of couple 13 is separated by Blast selected controls; pol sequences of couple 12 in phylogenetic tree is supported by a lower Bayesian posterior value.ConclusionParaphyly relationship between sequences of donator and recipient is only observed among partial HIV-1 positive couples with epidemiological link. Phylogenetic relationship is not always the same when various gene regions of HIV are used to conduct phylogenetic analysis. The combination of phylogenetic analysis based on various gene regions of HIV and enough epidemiology investigation is essential when inferring transmission direction of HIV in a transmission chain or in one couple. However, while observed paraphyly can be used to infer transmission direction in HIV-1 positive couple, no observed paraphyly cannot deny it.

HIV controllers suppress viral replication and evolution and prevent disease progression following intersubtype HIV-1 superinfection.

The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC). A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses. Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence. Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event. These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

Pretreatment HIV Drug Resistance and Virologic Outcomes to First-Line Antiretroviral Therapy in Peru.

Access to nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) first-line antiretroviral therapy (ART) for HIV has been increasing in Peru since a national ART program was initiated in 2004. Between 2007 and 2009, we found a 1% prevalence of pre-ART HIV drug resistance (PDR) among antiretroviral (ARV)-naive Peruvians. Given that PDR has been associated with virologic failure (VF) of ART, in 2014-2015 we enrolled a follow-up cohort at the same institution to determine whether the rate of transmitted resistance had increased and compared virologic outcomes of those with and without PDR. Blood specimens from ARV-naive individuals were assessed for PDR to NNRTI-based ART by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant within an individual's HIV quasispecies at reverse transcriptase codons M41L, K65R, K103N, Y181C, M184V, and G190A, and by Sanger consensus sequencing (CS). Rates of VF (plasma HIV RNA >200 copies/mL) were compared between those with and without PDR. Among 122 ARV-naive adults, PDR was detected by OLA in 17 (13.9%) adults. Compared with the 2007-2009 cohort, the proportion with PDR at OLA codons was significantly increased (p < .001). A total of 11 of 19 OLA mutations conferring high-level drug resistance were also detected by CS, and 8 additional participants had mutations encoding low-level resistance detected by CS for a total of 25 participants (20.5%). VF at month 6 of NNRTI-ART appeared greater in participants with versus without PDR [4/18 (22.2%) vs. 3/71 (4.2%); p = .03]. An increasing prevalence of PDR was detected among ARV-naive Peruvians. Studies are needed to determine risks of specific PDR mutations.