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1.  Genetic Diversity and Drug Resistance Mutations in HIV-1 from Untreated Patients in Niamey, Niger 
ISRN Microbiology  2011;2011:797463.
The objective of the study was to estimate the prevalence of transmitted resistance to antiretroviral of HIV-1 circulating in Niger. We collected plasmas from 96 drug-naive patients followed up in the main HIV/AIDS Care Center of Niamey, the capital city of Niger. After RNA extraction and retrotranscription to proviral DNA, nested PCR was performed to amplify PR (codons 1–99) and RT (codons 1–240) fragments for sequencing. Sequences were analysed for phylogeny, then for resistance-associated mutations according to IAS-USA and Stanford's lists of mutations. We characterized six HIV-1 genetic variants: CRF02-AG (56.3%), CRF30_0206 (15.6%), subtype G (15.6%), CRF06_cpx (9.4%), CRF11_cpx (2.1%), and CRF01_AE (1%). About 8.3% of HIV strains had at least 1 resistance mutation: 4 strains with at least 1 mutation to NRTI, 5 for NNRTI, and 1 for PI, respectiveley 4.2%, 5.2%, and 1.0%. These preliminary results gave enough information for the need of instauring HIV drug resistance national surveillance.
doi:10.5402/2011/797463
PMCID: PMC3658845  PMID: 23724311
2.  Recombinant Viruses Initiated the Early HIV-1 Epidemic in Burkina Faso 
PLoS ONE  2014;9(3):e92423.
We analyzed genetic diversity and phylogenetic relationships among 124 HIV-1 and 19 HIV-2 strains in sera collected in 1986 from patients of the state hospital in Ouagadougou, Burkina Faso. Phylogenetic analysis of the HIV-1 env gp41 region of 65 sequences characterized 37 (56.9%) as CRF06_cpx strains, 25 (38.5%) as CRF02_AG, 2 (3.1%) as CRF09_cpx, and 1 (1.5%) as subtype A. Similarly, phylogenetic analysis of the protease (PR) gene region of 73 sequences identified 52 (71.2%) as CRF06_cpx, 15 (20.5%) as CRF02_AG, 5 (6.8%) as subtype A, and 1 (1.4%) was a unique strain that clustered along the B/D lineage but basal to the node connecting the two lineages. HIV-2 PR or integrase (INT) groups A (n = 17 [89.5%]) and B (n = 2 [10.5%]) were found in both monotypic (n = 11) and heterotypic HIV-1/HIV-2 (n = 8) infections, with few HIV-2 group B infections. Based on limited available sampling, evidence suggests two recombinant viruses, CRF06_cpx and CRF02_AG, appear to have driven the beginning of the mid-1980s HIV-1 epidemic in Burkina Faso.
doi:10.1371/journal.pone.0092423
PMCID: PMC3960253  PMID: 24647246
3.  Identification and Characterization of CRF02_AG, CRF06_cpx, and CRF09_cpx Recombinant Subtypes in Mali, West Africa 
Abstract
Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to cocirculate in Africa. In West Africa, the high prevalence of CRF02_AG, and cocirculation of subtype A, CRF01_AE, CRF06_cpx, and other complex intersubtype recombinants has been well documented. Mali, situated in the heart of West Africa, is likely to be affected by the spread of recombinant subtypes. However, the dynamics of the spread of HIV-1 recombinant subtypes as well as nonrecombinant HIV-1 group M subtypes in this area have not been systematically assessed. Herein, we undertook genetic analyses on full-length env sequences derived from HIV-1-infected individuals living in the capital city of Mali, Bamako. Of 23 samples we examined, 16 were classified as CRF02_AG and three had a subsubtype A3. Among the remaining HIV-1 strains, CRF06_cpx and CRF09_cpx were each found in two patients. Comparison of phylogenies for six matched pol and full-length env sequences revealed that two strains had discordant subtype/CRF designations between the pol and env regions: one had A3polCRF02_AGenv and the other had CRF02_AGpolA3env. Taken together, our study demonstrated the high prevalence of CRF02_AG and complexity of circulating HIV-1 strains in Mali. It also provided evidence of ongoing virus evolution of CRF02_AG, as illustrated by the emergence of more complex CRF02_AG/A3 intersubtype recombinants in this area.
doi:10.1089/aid.2008.0111
PMCID: PMC2981380  PMID: 19182920
4.  Phylodynamics of the HIV-1 Epidemic in Cuba 
PLoS ONE  2013;8(9):e72448.
Previous studies have shown that the HIV-1 epidemic in Cuba displayed a complex molecular epidemiologic profile with circulation of several subtypes and circulating recombinant forms (CRF); but the evolutionary and population history of those viral variants remains unknown. HIV-1 pol sequences of the most prevalent Cuban lineages (subtypes B, C and G, CRF18_cpx, CRF19_cpx, and CRFs20/23/24_BG) isolated between 1999 and 2011 were analyzed. Maximum-likelihood analyses revealed multiple introductions of subtype B (n≥66), subtype C (n≥10), subtype G (n≥8) and CRF18_cpx (n≥2) viruses in Cuba. The bulk of HIV-1 infections in this country, however, was caused by dissemination of a few founder strains probably introduced from North America/Europe (clades BCU-I and BCU-II), east Africa (clade CCU-I) and central Africa (clades GCU, CRF18CU and CRF19CU), or locally generated (clades CRFs20/23/24_BG). Bayesian-coalescent analyses show that the major HIV-1 founder strains were introduced into Cuba during 1985–1995; whereas the CRFs_BG strains emerged in the second half of the 1990s. Most HIV-1 Cuban clades appear to have experienced an initial period of fast exponential spread during the 1990s and early 2000s, followed by a more recent decline in growth rate. The median initial growth rate of HIV-1 Cuban clades ranged from 0.4 year−1 to 1.6 year−1. Thus, the HIV-1 epidemic in Cuba has been a result of the successful introduction of a few viral strains that began to circulate at a rather late time of the AIDS pandemic, but then were rapidly disseminated through local transmission networks.
doi:10.1371/journal.pone.0072448
PMCID: PMC3767668  PMID: 24039765
5.  Analysis of the Origin and Evolutionary History of HIV-1 CRF28_BF and CRF29_BF Reveals a Decreasing Prevalence in the AIDS Epidemic of Brazil 
PLoS ONE  2011;6(3):e17485.
Background
HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28_BF and CRF29_BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear.
Methodology/Principal Findings
A collection of gag and pol sequences sampled within Brazil was screened for CRF28_BF-like and CRF29_BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28_BF-like and CRF29_BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988–1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31_BC.
Conclusions
We revealed that HIV-1 recombinants of the CRF28_BF/CRF29_BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.
doi:10.1371/journal.pone.0017485
PMCID: PMC3046974  PMID: 21390250
6.  Characterization of HIV-1 gag and nef in Cameroon: further evidence of extreme diversity at the origin of the HIV-1 group M epidemic 
Virology Journal  2013;10:29.
Background
Cameroon, in west central Africa, has an extraordinary degree of HIV diversity, presenting a major challenge for the development of an effective HIV vaccine. Given the continuing need to closely monitor the emergence of new HIV variants in the country, we analyzed HIV-1 genetic diversity in 59 plasma samples from HIV-infected Cameroonian blood donors. Full length HIV gag and nef sequences were generated and phylogenetic analyses were performed.
Findings
All gag and nef sequences clustered within HIV-1M. Circulating recombinant form CRF02_AG predominated, accounting for 50% of the studied infections, followed by clade G (11%), clade D and CRF37_cpx (4% each), and clades A, F, CRF01_AE and CRF36_cpx (2% each). In addition, 22% of the studied viruses apparently had nef and gag genes from viruses belonging to different clades, with the majority (8/10) having either a nef or gag gene derived from CRF02_AG. Interestingly, five gag sequences (10%) and three (5%) nef sequences were neither obviously recombinant nor easily classifiable into any of the known HIV-1M clades.
Conclusion
This suggests the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1M.
doi:10.1186/1743-422X-10-29
PMCID: PMC3560183  PMID: 23339631
HIV-1 diversity; West central Africa; RDP3; Maximum likelihood; PHYML
7.  Cross-Clade Recognition of HIV-1 CAp24 by CD4+ T Cells in HIV-1-Infected Individuals in Burkina Faso and Germany 
The Open AIDS Journal  2009;3:4-7.
The presence of antigen-specific cellular immune responses may be an indicator of long-term asymptomatic HIV-1-disease. The detection of cellular immune responses to infection with different subtypes of HIV-1 may be hampered by genetic differences of immunodominant antigens such as the capsid protein CAp24. In Nouna, Burkina Faso, HIV-1 circulating recombinant forms CRF02_AG and CRF06_cpx are the 2 major strains detectable in HIV-1-infected individuals, while subtype B strains prevail in Europe and North America. Amino acid sequences of CAp24 were assessed in blood samples from 10 HIV-1-infected patients in Nouna, Burkina Faso. Production of interferon-gamma (IFN-γ) in peripheral blood CD4+ lymphocytes in response to recombinant HIV-1 proteins derived from clade B (including CAp24NL4-3) was measured using a modified flow-cytometry-based whole blood short term activation assay (FASTimmune, BDBiosciences). IFN-γ production following stimulation with a whole length CAp24 protein derived from clade B (CAp24NL4-3) was additionally quantified in comparison to a CAp24 protein derived from CRF02_AG (CAp24BD6-15) in 16 HIV-1-infected patients in Heidelberg, Germany. Amino acid sequence identity of CAp24 obtained from patients in Nouna ranged between 86 and 89% when compared to the clade B CAp24NL4-3 consensus sequence, between 90 and 95% when compared to the circulating recombinant form CRF06_CPX consensus sequence, and between 92 and 96% when compared to the CAp24BD6-15 consensus sequence. Significant numbers of HIV-1-specific CD4+ lymphocytes producing IFN-γ were detected in 4 of 10 HIV-1-infected patients. In 7 of 16 patients in Heidelberg, recombinant CAp24BD6-15 stimulated IFN-γ-production in CD4+ lymphocytes to a similar extent as the clade B-derived CAp24NL4-3. Thus, antigen-specific CD4+ lymphocytes from both West African and European patients infected with different strains of HIV-1 show relevant cross-clade recognition of HIV-1 CAp24 in a flow-cytometry-based whole blood short term activation assay.
doi:10.2174/1874613600903010004
PMCID: PMC2701271  PMID: 19554213
8.  Emergence of a New HIV Type 1 CRF01_AE Variant in Guangxi, Southern China 
AIDS Research and Human Retroviruses  2012;28(10):1352-1356.
Abstract
The distribution of HIV-1 subtypes and genetic characterization of CRF01_AE in Guangxi, southern China were identified. The distribution of HIV-1 genotypes based on gag, pol, and partial env sequences (n=349) was as follows: CRF01_AE (66.5%), CRF08_BC (19.2%), CRF07_BC (7.2%), URF (4.6%), subtype B (1.7%), and subtype B′ (0.9%). CRF01_AE predominated in all geographic regions and risk populations and there were multiple introductions of CRF01_AE strains in Guangxi. We found a peculiar CRF01_AE monophyletic lineage distinct from other CRF01_AE viruses, and we designated it “CRF01_AE-v” for convenience. CRF01_AE-v circulating in both heterosexuals and injecting drug users (IDUs) had accounted for 39.7% of CRF01_AE. It showed a selective advantage in the Guangxi population and formed its own characteristic compared with all the CRF01_AE references. Our results suggested that CRF01_AE-v was a new variant of CRF01_AE and it might lead to a new epidemic in Guangxi.
doi:10.1089/aid.2011.0364
PMCID: PMC3448111  PMID: 22264007
9.  Temporal and Spatial Dynamics of Human Immunodeficiency Virus Type 1 Circulating Recombinant Forms 08_BC and 07_BC in Asia▿ † 
Journal of Virology  2008;82(18):9206-9215.
Human immunodeficiency virus type 1 (HIV-1) CRF08_BC and CRF07_BC are two major recombinants descended from subtypes B′ and C. Despite their massive epidemic impact in China, their migration patterns and divergence times remain unknown. Phylogenetic and population genetic analyses were performed on 228 HIV-1 sequences representing CRF08_BC, CRF07_BC, and subtype C strains from different locations across China, India, and Myanmar. Genome-specific rates of evolution and divergence times were estimated using a Bayesian Markov chain Monte Carlo framework under various evolutionary models. CRF08_BC originated in 1990.3 (95% credible region [CR], 1988.6 to 1991.9) in Yunnan province before spreading to Guangxi (south) and Liaoning (northeast) around 1995. Inside Guangxi region, the eastward expansion of CRF08_BC continued from Baise city (west) to Binyang (central) between 1997 and 1998 and later spread into Pingxiang around 1999 in the south, mainly through injecting drug users. Additionally, CRF07_BC diverged from its common ancestor in 1993.3 (95% CR, 1991.2 to 1995.2) before crossing the border into southern Taiwan in late 1990s. Phylogenetic analysis indicates that both CRF08_BC and CRF07_BC can trace their origins to Yunnan. The parental Indian subtype C lineage likely entered China around 1981.2 (95% CR, 1976.7 to 1985.9). Using a multiple unlinked locus model, we also showed that the dates of divergence calculated in this study may not be significantly affected by intrasubtype recombination among different lineages. This is the first phylodynamic study depicting the spatiotemporal dynamics of HIV/AIDS in East Asia.
doi:10.1128/JVI.00399-08
PMCID: PMC2546895  PMID: 18596096
10.  Analysis of pol Integrase Sequences in Diverse HIV Type 1 Strains Using a Prototype Genotyping Assay 
Abstract
A prototype assay was used to genotype integrase (IN) from 120 HIV-1- infected IN inhibitor-naive adults from Argentina, Brazil, Cameroon, South Africa, Thailand, and Uganda. Subtype designations based on analysis of pol IN sequences were A (14), B (15), C (12), D (11), F (12), G (7), H (1), CRF01_AE (9), CRF02_AG (34), CRF22_01A1 (4), and CRF37_cpx (1). Ten (8.3%) of 120 samples had mutations associated with reduced susceptibility to the IN inhibitors, raltegravir and elvitegravir. Two samples had E92Q (both subtype B) and eight had E157Q (2A, 1C, 1D, 1F, 3 CRF02_AG). Some samples had other mutations selected by these drugs including T97A, and some had amino acid polymorphisms at positions associated with raltegravir and elvitegravir resistance. Mutations associated with other investigational HIV IN inhibitors were also identified. This suggests that HIV strains may vary in their natural susceptibility to HIV IN inhibitors.
doi:10.1089/aid.2008.0236
PMCID: PMC2853838  PMID: 19327053
11.  The Evolution of HIV-1 Diversity in Rural Cameroon and its Implications in Vaccine Design and Trials 
Viruses  2010;2(2):639-654.
West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (~40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.
doi:10.3390/v2020639
PMCID: PMC2975583  PMID: 21072143
HIV-1 Diversity; Rural Cameroon; phylogenetics
12.  The Evolution of HIV-1 Diversity in Rural Cameroon and its Implications in Vaccine Design and Trials 
Viruses  2010;2(2):639-654.
West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (∼40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.
doi:10.3390/v2020639
PMCID: PMC2975583  PMID: 21072143
HIV-1 Diversity; Rural Cameroon; phylogenetics
13.  Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial 
Journal of Virology  2013;87(13):7265-7281.
The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.
doi:10.1128/JVI.03070-12
PMCID: PMC3700312  PMID: 23576510
14.  Genetic architecture of HIV-1 genes circulating in north India & their functional implications 
This review presents data on genetic and functional analysis of some of the HIV-1 genes derived from HIV-1 infected individuals from north India (Delhi, Punjab and Chandigarh). We found evidence of novel B/C recombinants in HIV-1 LTR region showing relatedness to China/Mynmar with 3 copies of Nfκb sites; B/C/D mosaic genomes for HIV-1 Vpr and novel B/C Tat. We reported appearance of a complex recombinant form CRF_02AG of HIV-1 envelope sequences which is predominantly found in Central/Western Africa. Also one Indian HIV-1 envelope subtype C sequence suggested exclusive CXCR4 co-receptor usage. This extensive recombination, which is observed in about 10 per cent HIV-1 infected individuals in the Vpr genes, resulted in remarkably altered functions when compared with prototype subtype B Vpr. The Vpu C was found to be more potent in causing apoptosis when compared with Vpu B when analyzed for subG1 DNA content. The functional implications of these changes as well as in other genes of HIV-1 are discussed in detail with possible implications for subtype-specific pathogenesis highlighted.
doi:10.4103/0971-5916.92624
PMCID: PMC3284088  PMID: 22310812
HIV-1 genetic variants; HIV-1 recombinants; HIV-1 subtypes; India
15.  Detailed Molecular Epidemiologic Characterization of HIV-1 Infection in Bulgaria Reveals Broad Diversity and Evolving Phylodynamics 
PLoS ONE  2013;8(3):e59666.
Limited information is available to describe the molecular epidemiology of HIV-1 in Bulgaria. To better understand the genetic diversity and the epidemiologic dynamics of HIV-1 we analyzed 125 new polymerase (pol) sequences from Bulgarians diagnosed through 2009 and 77 pol sequences available from our previous study from persons infected prior to 2007. Epidemiologic and demographic information was obtained from each participant and phylogenetic analysis was used to infer HIV-1 evolutionary histories. 120 (59.5%) persons were infected with one of five different HIV-1 subtypes (A1, B, C, F1 and H) and 63 (31.2%) persons were infected with one of six different circulating recombinant forms (CRFs; 01_AE, 02_AG, 04_cpx, 05_DF, 14_BG, and 36_cpx). We also for the first time identified infection with two different clusters of unique A-like and F-like sub-subtype variants in 12 persons (5.9%) and seven unique recombinant forms (3.5%), including a novel J/C recombinant. While subtype B was the major genotype identified and was more prevalent in MSM and increased between 2000–2005, most non-B subtypes were present in persons ≥45 years old. CRF01_AE was the most common non-B subtype and was higher in women and IDUs relative to other risk groups combined. Our results show that HIV-1 infection in Bulgaria reflects the shifting distribution of genotypes coincident with the changing epidemiology of the HIV-1 epidemic among different risk groups. Our data support increased public health interventions targeting IDUs and MSM. Furthermore, the substantial and increasing HIV-1 genetic heterogeneity, combined with fluctuating infection dynamics, highlights the importance of sustained and expanded surveillance to prevent and control HIV-1 infection in Bulgaria.
doi:10.1371/journal.pone.0059666
PMCID: PMC3602066  PMID: 23527245
16.  Impact of HIV Type 1 Subtype on Drug Resistance Mutations in Nigerian Patients Failing First-Line Therapy 
Abstract
A diverse array of non-subtype B HIV-1 viruses circulates in Africa and dominates the global pandemic. It is important to understand how drug resistance mutations in non-B subtypes may develop differently from the patterns described in subtype B. HIV-1 reverse transcriptase and protease sequences from 338 patients with treatment failure to first-line ART regimens were evaluated. Multivariate logistic regression was used to examine the effect of subtype on each mutation controlling for regimen, time on therapy, and total mutations. The distribution of HIV-1 subtypes included CRF02_AG (45.0%), G (37.9%), CRF06_cpx (4.4%), A (3.6%), and other subtypes or recombinant sequences (9.2%). The most common NRTI mutations were M184V (89.1%) and thymidine analog mutations (TAMs). The most common NNRTI mutations were Y181C (49.7%), K103N (36.4%), G190A (26.3%), and A98G (19.5%). Multivariate analysis showed that CRF02_AG was less likely to have the M41L mutation compared to other subtypes [adjusted odds ratio (AOR) = 0.35; p = 0.022]. Subtype A patients showed a 42.5-fold increased risk (AOR = 42.5, p = 0.001) for the L210W mutation. Among NNRTI mutations, subtype G patients had an increased risk for A98G (AOR = 2.40, p = 0.036) and V106I (AOR = 6.15, p = 0.010), whereas subtype CRF02_AG patients had an increased risk for V90I (AOR = 3.16; p = 0.003) and a decreased risk for A98G (AOR = 0.48, p = 0.019). Five RT mutations were found to vary significantly between different non-B West African subtypes. Further study to understand the clinical impact of subtype-specific diversity on drug resistance will be critically important to the continued success of ART scale-up in resource-limited settings.
doi:10.1089/aid.2010.0050
PMCID: PMC3034099  PMID: 20964479
17.  Near Full-Length Sequence Analysis of HIV Type 1 BF Recombinants from Italy 
Abstract
Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance.
doi:10.1089/aid.2011.0002
PMCID: PMC3292755  PMID: 21740272
18.  Molecular Diversity of HIV-1 among People Who Inject Drugs in Kuala Lumpur, Malaysia: Massive Expansion of Circulating Recombinant Form (CRF) 33_01B and Emergence of Multiple Unique Recombinant Clusters 
PLoS ONE  2013;8(5):e62560.
Since the discovery of HIV-1 circulating recombinant form (CRF) 33_01B in Malaysia in the early 2000 s, continuous genetic diversification and active recombination involving CRF33_01B and other circulating genotypes in the region including CRF01_AE and subtype B′ of Thai origin, have led to the emergence of novel CRFs and unique recombinant forms. The history and magnitude of CRF33_01B transmission among various risk groups including people who inject drugs (PWID) however have not been investigated despite the high epidemiological impact of CRF33_01B in the region. We update the most recent molecular epidemiology of HIV-1 among PWIDs recruited in Malaysia between 2010 and 2011 by population sequencing and phylogenetic analysis of 128 gag-pol sequences. HIV-1 CRF33_01B was circulating among 71% of PWIDs whilst a lower prevalence of other previously dominant HIV-1 genotypes [subtype B′ (11%) and CRF01_AE (5%)] and CRF01_AE/B′ unique recombinants (13%) were detected, indicating a significant shift in genotype replacement in this population. Three clusters of CRF01_AE/B′ recombinants displaying divergent yet phylogenetically-related mosaic genomes to CRF33_01B were identified and characterized, suggestive of an abrupt emergence of multiple novel CRF clades. Using rigorous maximum likelihood approach and the Bayesian Markov chain Monte Carlo (MCMC) sampling of CRF33_01Bpol sequences to elucidate the past population dynamics, we found that the founder lineages of CRF33_01B were likely to have first emerged among PWIDs in the early 1990 s before spreading exponentially to various high and low-risk populations (including children who acquired infections from their mothers) and later on became endemic around the early 2000 s. Taken together, our findings provide notable genetic evidence indicating the widespread expansion of CRF33_01B among PWIDs and into the general population. The emergence of numerous previously unknown recombinant clades highlights the escalating genetic complexity of HIV-1 in the Southeast Asian region.
doi:10.1371/journal.pone.0062560
PMCID: PMC3646884  PMID: 23667490
19.  Characterization of Primary Isolates of HIV Type 1 CRF28_BF, CRF29_BF, and Unique BF Recombinants Circulating in São Paulo, Brazil 
Abstract
We report for the first time the genetic and biological characterization of 10 HIV-1 primary isolates representing CRF28_BF and CRF29_BF together with additional unique BF recombinant forms (URFs) obtained by PBMC cocultivation. Recombination is an important factor promoting the increase in the genetic diversity of HIV-1. Notably, more than 20% of HIV-1 sequences worldwide were recombinants. Several recombinant viruses were reported in Brazil, and six circulating recombinant forms (CRFs) have been identified (CRF28_BF, CRF29_BF, CRF31_BC, CRF39_BF, CRF40_BF, and CRF46_BF). CRF28_BF and CRF29_BF were found to infect almost 30% of the patients in São Paulo State. The near full-length genomes of these 10 primary isolates were amplified by nested PCR in three overlapping segments, purified, and sequenced. Three samples were related to CRF28_BF, three to CRF29_BF, and four were unique recombinant forms (URFs), as determined by their breakpoint profile determined with the jpHMM program. Additionally, the coreceptor usage of these isolates was investigated in vitro using GHOST assays, which revealed three dual-tropic (X4/R5) viruses, four lymphotropic (X4) viruses, and three macrophage-tropic (R5) viruses with different V3-loop motifs, which challenges the notion that GWGR-carrying viruses are macrophage-tropic only. In sum, we report a much-anticipated well-characterized panel of viruses representing CRF28_BF, CRF29_BF, and URFs from São Paulo State, Brazil.
doi:10.1089/aid.2011.0123
PMCID: PMC3423645  PMID: 22176121
20.  New and old complex recombinant HIV-1 strains among patients with primary infection in 1996–2006 in France: The French ANRS CO06 primo cohort study 
Retrovirology  2008;5:69.
Background
Prevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs).
Methods
Among 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures.
Results
Among V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006–2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France.
Conclusion
This study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity.
doi:10.1186/1742-4690-5-69
PMCID: PMC2553414  PMID: 18673538
21.  Identifying the Important HIV-1 Recombination Breakpoints 
PLoS Computational Biology  2008;4(9):e1000178.
Recombinant HIV-1 genomes contribute significantly to the diversity of variants within the HIV/AIDS pandemic. It is assumed that some of these mosaic genomes may have novel properties that have led to their prevalence, particularly in the case of the circulating recombinant forms (CRFs). In regions of the HIV-1 genome where recombination has a tendency to convey a selective advantage to the virus, we predict that the distribution of breakpoints—the identifiable boundaries that delimit the mosaic structure—will deviate from the underlying null distribution. To test this hypothesis, we generate a probabilistic model of HIV-1 copy-choice recombination and compare the predicted breakpoint distribution to the distribution from the HIV/AIDS pandemic. Across much of the HIV-1 genome, we find that the observed frequencies of inter-subtype recombination are predicted accurately by our model. This observation strongly indicates that in these regions a probabilistic model, dependent on local sequence identity, is sufficient to explain breakpoint locations. In regions where there is a significant over- (either side of the env gene) or under- (short regions within gag, pol, and most of env) representation of breakpoints, we infer natural selection to be influencing the recombination pattern. The paucity of recombination breakpoints within most of the envelope gene indicates that recombinants generated in this region are less likely to be successful. The breakpoints at a higher frequency than predicted by our model are approximately at either side of env, indicating increased selection for these recombinants as a consequence of this region, or at least part of it, having a tendency to be recombined as an entire unit. Our findings thus provide the first clear indication of the existence of a specific portion of the genome that deviates from a probabilistic null model for recombination. This suggests that, despite the wide diversity of recombinant forms seen in the viral population, only a minority of recombination events appear to be of significance to the evolution of HIV-1.
Author Summary
Multiple variants of HIV can infect the same cell, and because each viral particle contains two copies of the viral genomic RNA, RNAs from different viruses can occasionally be incorporated together within a viral particle. When this virus subsequently infects another cell, genetic exchange (recombination) may occur between these two divergent copies of genomic RNA as a result of a switch between the RNA molecules while they are copied into DNA. This process is very important to understand as it contributes to the generation of new HIV variants. In this study, we have analysed a set of recombinant HIV genomes generated in the laboratory to construct a probabilistic model of the propensity for the switch to take place in specific regions of the genome, dependent on the local similarity of the parental viral sequences. This model allows us to predict the locations where recombination should occur more frequently. By comparing these predictions to the patterns of recombination observed in the HIV-1 pandemic, we identify the genomic regions in which recombination has been more important, in that it has provided an evolutionary advantage to the virus.
doi:10.1371/journal.pcbi.1000178
PMCID: PMC2522274  PMID: 18787691
22.  Characterization of Acute HIV-1 Infection in High-Risk Nigerian Populations 
The Journal of Infectious Diseases  2012;205(8):1239-1247.
Background. Acute phase of human immunodeficiency virus (HIV) infection (AHI) may account for a significant proportion of HIV-1 transmission. We identified and characterized individuals in Nigeria with AHI.
Methods. Individuals were tested using a combination of rapid HIV testing in mobile units and laboratory-based specimen pooling for nucleic acid amplification testing. Genome sequences were characterized. A linear segmented regression model was fit to serial viral load (VL) measurements to characterize early VL profiles.
Results. Sixteen AHIs were identified from 28 655 persons screened. Specimens were genotyped: 7 (43.8%) were CRF02_AG, 6 (37.5%) were subtype G, 1 (6.3%) was CRF06_cpx, and 2 (12.5%) were unique recombinant forms. No antiretroviral resistance mutations were detected. The mean duration of high VL burden from peak to nadir was 76 days (95% confidence interval [CI], 58–93 days), and the mean rate of viremic control was −0.66 log10 VL per month. The mean VL at set-point was 4.5 log10 copies/mL (95% CI, 3.9–5.1 log10 copies/mL).
Conclusions. This study is the first to characterize AHI among Nigerians identified as HIV infected before seroconversion who would be otherwise missed by conventional HIV testing. Infections by HIV subtypes in Nigeria exhibit long periods of high viral burden, which can contribute to increased transmissibility.
doi:10.1093/infdis/jis103
PMCID: PMC3308903  PMID: 22357663
23.  Most HIV Type 1 Non-B Infections in the Spanish Cohort of Antiretroviral Treatment-Naïve HIV-Infected Patients (CoRIS) Are Due to Recombinant Viruses 
Journal of Clinical Microbiology  2012;50(2):407-413.
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.
doi:10.1128/JCM.05798-11
PMCID: PMC3264155  PMID: 22162552
24.  Current Trends of HIV Recombination Worldwide 
Infectious Disease Reports  2013;5(Suppl 1):e4.
One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O), as well as interand intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs) and unique recombinant forms (URFs), requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and cocirculation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.
doi:10.4081/idr.2013.s1.e4
PMCID: PMC3892622  PMID: 24470968
HIV; current trends
25.  Characterization of full-length HIV-1 CRF17_BF genomes and comparison to the prototype CRF12_BF strains ☆ 
The aim of this work is to characterize the full-length intersubtype recombinant structure of the HIV-1 Circulating Recombinant Form CRF17_BF. A single genome of CRF17_BF was originally described in 2001 as being largely similar to CRF12_BF. Since then, more genomes of CRF17_BF have been sequenced but not adequately described in publications. Here we describe CRF17_BF as a genuine CRF, and analyze its recombination pattern based on bootscan analyses, subtype signature patterns, and phylogenetic reconstruction of subtype-delimited segments. We show that CRF17_BF can be distinguished from CRF12_BF in several regions of the genome, including vpu, pol, env and nef. A complete and accurate characterization and description of recombination breakpoints in CRFs is required for a proper surveillance of HIV-1 genotypes, and important for epidemiological purposes.
doi:10.1016/j.meegid.2012.01.003
PMCID: PMC3471996  PMID: 22266022
HIV-1 subtypes; Argentina; BF recombinants; HIV diversity; Molecular epidemiology; CRF17_BF

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