AG Recombinants Research Articles

New Subtype B Containing HIV-1 Circulating Recombinant of sub-Saharan Africa Origin in Nigerian Men Who Have Sex With Men.

HIV-1 circulating recombinant forms (CRF) containing subtype B are uncommon in sub-Saharan Africa. Prevalent infections observed during enrollment of a prospective study of men who have sex with men (MSM) from Lagos, Nigeria, revealed the presence of a family of subtype B and CRF02_AG recombinants. This report describes the HIV-1 genetic diversity within a high-risk, high-prevalence, and previously undersampled cohort of Nigerian MSM. Between 2013 and 2016, 672 MSM were enrolled at the Lagos site of the TRUST/RV368 study. Prevalent HIV-1 infections were initially characterized by pol sequencing and phylogenetic subtyping analysis. Samples demonstrating the presence of subtype B were further characterized by near full-length sequencing, phylogenetic, and Bayesian analyses. Within this cohort, HIV-1 prevalence was 59%. The major subtype was CRF02_AG (57%), followed by CRF02/B recombinants (15%), subtype G (13%), and smaller amounts of A1, B, and other recombinants. Nine clusters of closely related pol sequences indicate ongoing transmission events within this cohort. Among the CRF02_AG/B, a new CRF was identified and termed CRF95_02B. Shared risk factors and Bayesian phylogenetic inference of the new CRF95_02B and the similarly structured CRF56_cpx indicate a Nigerian or West African origin of CRF56_cpx before its observation in France. With high HIV-1 prevalence, new strains, and multiple transmission networks, this cohort of Nigerian MSM represents a previously hidden reservoir of HIV-1 strains, including the newly identified CRF95_02B and closely related CRF56_cpx. These strains will need to be considered during vaccine selection and development to optimize the design of a globally effective HIV-1 vaccine.

Pre-treatment drug resistance and HIV-1 subtypes in infants from Argentina with and without exposure to antiretroviral drugs for prevention of mother-to-child transmission.

To assess the prevalence and patterns of pre-treatment HIV drug resistance (PDR) and HIV-1 subtype in infants from Argentina with exposure to different antiretroviral drugs (ARVs) for the prevention of mother-to-child transmission (PMTCT). HIV-1 genotyping was performed in 115 infants (median age = 2.3 months) born between 2007 and 2014 to screen for drug resistance mutations (DRMs) before starting first-line ART. HIV-1 subtype was characterized by phylogenetic and recombination analysis. Overall, DRMs were found in 34 of 115 infants (PDR level 30% to any ARV, 3.5% to PIs, 12% to NRTIs and 22% to NNRTIs). Of the 115 infants, 22 (19.1%) were ARV-unexposed. Another 93 were ARV-exposed: 28 (24.3%) to short-course zidovudine monotherapy ARV prophylaxis; 25 (21.7%) to nevirapine-based ARV prophylaxis; 12 (10.4%) to perinatal infant zidovudine prophylaxis + maternal combination ART with NNRTIs; and 28 (24.3%) to perinatal infant zidovudine prophylaxis+maternal combination ART with PIs. Transmitted drug resistance among ARV-unexposed infants was 32% (5% to PIs, 9% to NRTIs and 18% to NNRTIs). ART-exposed infants showed multi-class ARV resistance. Importantly, vertical transmission of a triple-class-resistant virus was confirmed in one case. Patterns of DRMs predicted high-level resistance to NNRTIs in a similar and high proportion (>50%) of infants with at least one DRM independently of ARV exposure. BF recombinants were found in 74%, subtype B in 20%, subtype C in 3% and novel AG and AB recombinants in 3%. PDR in HIV-1-infected children from Argentina is among the highest reported, jeopardizing successful lifelong suppressive ART as well as the efficacy of current PMTCT regimens.

Increase in human immunodeficiency virus 1 diversity and detection of various subtypes and recombinants in north-eastern Brazil.

Diverse human immunodeficiency virus 1 (HIV-1) subtypes and circulating recombinant forms are found in Brazil. The majority of HIV-1 molecular epidemiological studies in Brazil have been conducted in the southern and south-eastern regions of the country, although several recent studies in the north-eastern region have addressed this issue. The objective of this study was to molecularly characterize HIV-1 circulating in Pernambuco, north-eastern Brazil. A total of 64 samples were collected from 2002 to 2003, and another 103 were collected from 2007 to 2009. The protease and partial reverse transcriptase regions of the HIV-1 polymerase-encoding (pol) gene were sequenced, and subtyping, recombination and phylogenetic analyses were performed.Results/Key findings. Subtype B (60.9 %) was found to be predominant, followed by HIV-1 F (31.4 %). Several BF recombinants (4.2 %), and BC and AG recombinants were also identified. The intra-subtype genetic diversity was estimated to be 0.065 (sd±0.004) for HIV-1 B and 0.055 (sd±0.004) for HIV-1 F, reflecting a greater accumulation of mutations in subtype B (P<0.01). More codons were found to be under positive selective pressure in samples collected from 2007 to 2009, from individuals with a T-cell count≥200 cells mm-3 and from women. Coalescence data indicated that the subtype F population has been continuously expanding. HIV-1 shows high genetic diversity in the state of Pernambuco. Thus, additional molecular evaluations of circulating strains will provide a better understanding of the epidemic and may lead to more effective preventive strategies.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG.

While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.The sensitivity and specificity of MHA varied between 73-100% and 90-100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.

High HIV-1 Diversity and Prevalence of Transmitted Drug Resistance Among Antiretroviral-Naive HIV-Infected Pregnant Women from Rio de Janeiro, Brazil.

Antiretroviral (ARV) resistance mutations in human immunodeficiency virus type 1 (HIV-1) infection may reduce the efficacy of prophylactic therapy to prevent mother-to-child transmission (PMTCT) and future treatment options. This study evaluated the diversity and the prevalence of transmitted drug resistance (TDR) in protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene among 87 ARV-naive HIV-1-infected pregnant women from Rio de Janeiro, Brazil, between 2012 and 2015. The viral diversity comprised HIV-1 subtypes B (67.8%), F1 (17.2%), and C (4.6%); the circulating recombinant forms 12_BF (2.3%), 28/29_BF, 39_BF, 02_AG (1.1% each) and unique recombinants forms (4.5%). The overall prevalence of any TDR was 17.2%, of which 5.7% for nucleoside RT inhibitors, 5.7% for non-nucleoside RT inhibitors, and 8% for PR inhibitors. The TDR prevalence found in this population may affect the virological outcome of the standard PMTCT ARV-regimens, reinforcing the importance of continuous monitoring.

The Genetic Variability of HIV-1 in Kyrgyzstan: The Spread of CRF02_AG and Subtype A1 Recombinants

The Genetic Variability of HIV-1 in Kyrgyzstan: The Spread of CRF02_AG and Subtype A1 Recombinants

HIV-1 subtype distribution determined by phylogenetic analysis of pol gene sequences and automated subtyping tools among HIV-1 isolates from the Aegian Region of Turkey

Human immunodeficiency virus (HIV) exhibiting remarkable genetic variability, includes two genotypes namely HIV-1 (group M, N, O and P) and HIV-2 (group A-H). HIV-1 group M, which is mainly the cause of the AIDS pandemic, is divided into nine pure subtypes, more than 45 circulating recombinant forms (CRF) and numerous unique recombinant forms (URF). According to the documents of Turkish Government of Health, among a total of 6802 HIV-positive cases, 1096 of them were defined as AIDS as of June 2013 in Turkey. Although subtype B is the predominant subtype, recent studies indicate higher proportion of CRFs similar to their increasing role in the HIV pandemic. The aim of this study was to determine the subtype distribution of HIV-1 strains isolated from 70 patients (61 male, 9 female; age range: 16-73 yrs, mean age: 39.6 yrs) who presented to our institution between April 2008-June 2013. HIV-1 strains were subtyped by phylogenetic analysis of the pol gene region and commonly used automated subtyping tools namely, Stanford HIV db v6.2.0 and Rega v3.0. Pol sequences retrieved from the Los Alamos database and from GeneBank, were trimmed from full-length genomes. Phylogenetic analysis of the 1302 base pair of the pol gene region was performed using Mega v5.2 software. The sequences were aligned using Muscle and phylogenetic distances between sequences were estimated by using Kimura two-parameter model (transition/transversion ratio: 2.0). Tree topology was obtained using neighbour-joining method and bootstrap value was set at 1000. Sixty-one (87.1%) patients were antiretroviral treatment (ART)-naive and nine were on different ART regimens. The subtypes of the isolates according to phylogenetic analysis were found as follows; 31 (44.2%) subtype B, 24 (34.2%) CRF42_BF, 6 (8.5%) B/CRF02_AG recombinants, 5 (7.1%) sub-subtype A1, 1 (1.4%) sub-subtype F1, 1 (%1.4) CRF 25_cpx, 1 (1.4%) CRF02_AG and 1 (1.4%) CRF01_AE. Rega v3.0 subtyping tool produced five discrepant results (4 B/CRF02-AG and 1 CRF42_BF) compared to phylogenetic analysis. Stanford HIVdb v6.2.0 had eight results (3 CRF42_BF, 2 subtype B, 2 sub-subtype A1, 1 CRF25_cpx) that were not concordant with phylogenetic analysis. Stanford HIVdb v6.2.0 was able to subtype all B/CRF02_AG recombinant strains. B/CRF02_AG recombinants which were seen among homosexual men in France were for the first time isolated in Turkey from five men (2 homosexual, 2 bisexual, 1 heterosexual) and one heterosexual woman. CRF42_BF had not been found in Turkey previously and it has not been a common type isolated in neighboring countries either. Full genome sequencing could be helpful to further analysis of those isolates. Our results support the latest studies from Turkey reporting increase in the proportion of CRF-related infections. This is not an unusual finding when geographical location of Turkey is considered. Nevertheless, more comprehensive data regarding molecular epidemiology and subtype distribution of HIV-1 isolates in Turkey are needed.

Most HIV Type 1 Non-B Infections in the Spanish Cohort of Antiretroviral Treatment-Naïve HIV-Infected Patients (CoRIS) Are Due to Recombinant Viruses

HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected.

Identification of new, emerging HIV-1 unique recombinant forms and drug resistant viruses circulating in Cameroon

BackgroundThe HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients.MethodologyBlood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database.ResultsPhylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies.ConclusionsOur results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.

The Changing Trend of HIV Type 1 Subtypes in Nairobi

Monitoring the distribution of HIV-1 subtypes and recombinants among infected individuals has become a priority in HIV therapy. A laboratory analysis of samples collected from HIV-positive patients attending an STI clinic in Nairobi was done between March and May 2004. PCR was carried out on pol (intergrase) and env (C2V3) regions and resulting data on the 54 samples successfully analyzed revealed the following as circulating subtypes: 35/54(65%) were A1/A1, 5/54(9%) were A/C, 4/54 (7%) were A1/D, 1/54 (2%) was C/D, 1/54 (2%) was D/D, 1/54 (2%) was A1/A2, 1/54 (2%)was G/G, 1/54 (2%) was A2/D, 1/54 (2%) was C/C, and 4/54 (7%) were CRF02_ AG. The results show an increase in HIV-1 recombinants with the emergence of A1/A2 and an increase in CRF02_AG recombinants. Subtype diversity in the advent of ARV use will impact negatively on treatment outcomes. As such, increased viral evolution and recombination will call for continuous evaluation of available anti-HIV regimens for better management of those infected with HIV-1.

Quasispecies analysis of novel HIV‐1 recombinants of subtypes A and G reveals no similarity to the mosaic structure of CRF02_AG

HIV-1 circulating recombinant form (CRF) 02_AG is responsible for greater than 65% of HIV-1 infections in Cameroon and is widespread across West and West-Central Africa. The parental subtypes A1 and G cocirculate in this part of Africa, and high rates of infection predispose to the generation of AG unique recombinant forms (URFs). Little is known as to whether A1 and G can recombine and thrive in vivo with breakpoints other than those characteristic of CRF02_AG. In this study, six unique recombinant viruses of subtypes A1 and G were identified in two individuals in Cameroon. A 1.5 kb fragment of the reverse transcriptase (RT) region of pol (HXB2 location 2,612-4,159) and the entire env gene (HXB2 location 6,202-9,096) were evaluated by phylogenetic and breakpoint analyses. Each URF was found to have breakpoints different than CRF02_AG, indicating that A and G gene segments are functionally compatible with more than one pattern of recombination. Furthermore, contemporaneous, cultured viruses from these individuals were analyzed, revealing different proportions of URFs compared to those found in plasma, possibly indicating compart mentalization and/or phenotypic variation among the URFs. CRF02_AG emerged from West-Central Africa to become a highly successful viral strain. As such, monitoring the spread of newly emerging AG recombinants is critical not only for understanding the epidemiology of HIV-1, but also in the design of future therapeutics and vaccines appropriate to this part of Africa, and globally.

HIV‐1 subtypes in Spain: a retrospective analysis from 1995 to 2003

To perform a retrospective analysis of all HIV-1 non-B variants circulating in Spain from 1995 to 2003 and extend their virological characterization. Samples from a total of 396 HIV-infected subjects with epidemiological suspicion of being infected with non-B clades were analysed during the study period. Subtyping was carried out on the protease (PR), reverse transcriptase (RT) and envelope (env) genes. PR sequences belonging to non-B subtypes were recognized in 43.2% of cases (23 A, 13C, 6D, 3F, 118 G, 3H, 4 J and 1 U). Subtype G and AG recombinants were the most frequent variants (69%), and were found most often in subjects from West and Central Africa. Up to 70% of pol (PR, RT) sequences belonging to subtype G harboured env sequences belonging to clade A (55%), B (13.8%) or K (3.4%). Nearly half were mosaic GA viruses, and a few were CRF 14 BG viruses. Up to 14 new recombinant viruses, which could not be assigned to previously described circulating recombinant forms (CRFs), were found. There is great diversity in the HIV-1 variants and recombinant viruses circulating in Spain. Non-B sequences may be underestimated if only the env region is examined in phylogenetic analyses. Drug resistance testing provides the advantage of pol subtyping, and its additional use for this purpose should be encouraged.

The AG Recombinant IbNG and Novel Strains of Group M HIV-1 Are Common in Cameroon

The genetic diversity of group M HIV-1 is highest in west central Africa. Blood samples from four locations in Cameroon were collected to determine the molecular epidemiology of HIV-1. The C2-V5 region of envelope was sequenced from 39 of the 40 samples collected, and 7 samples were sequenced across the genome. All strains belonged to group M of HIV-1. The circulating recombinant form CRF02 AG (IbNG) was the most common strain (22/39, 56%). Two of these were confirmed by full genome analysis. Four samples (4/39, 10%) clustered with the sub-subtype F2 and one of these was confirmed by full genome sequencing. Recombinant forms, each different but containing subtype A, accounted for the next most common form (7/39, 18%). Among these recombinants, those combining subtypes A and G were the most common (4/7, 57%). Also found were 3 subtype A, 2 subtype G, and 1 subtype B strain. Many recombination break points were shared between IbNG and the other AG recombinants, though none of these other AG recombinants included IbNG as a parent. This suggests that there was an ancestral AG recombinant that gave rise to CRF02 AG (IbNG), the successful circulating recombinant form, and to others that were less successful and are now rare.

Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa.

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A(gag)/G(pol)/A(env), three with A(gag)/G(pol)/G(env) and one other with A(gag) /G(pol)/G(env), in addition to 'pure' subtypes A(gag)/A(pol)/A(env) (n=1) and G(gag)/G(pol)/G(env) (n=4). To analyse the crossover points in more detail, a new RT-PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A(gag)/G(pol)/A(env) recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A(gag)/G(pol) /G(env) recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.