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International audience; Background: Global HIV-1 genetic diversity and evolution form a major challenge to treatment and prevention efforts. An increasing number of distinct HIV-1 recombinants have been identified worldwide, but their contribution to the global epidemic is unknown. We aimed to estimate the global and regional distribution of HIV-1 recombinant forms during 1990-2015.Methods: We assembled a global HIV-1 molecular epidemiology database through a systematic literature review and a global survey. We searched the PubMed, Embase (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) databases for HIV-1 subtyping studies published from Jan 1, 1990, to Dec 31, 2015. Unpublished original HIV-1 subtyping data were collected through a survey among experts in the field who were members of the WHO-UNAIDS Network for HIV Isolation and Characterisation. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. Countries were grouped into 14 regions and analyses were done for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV in each country to generate regional and global estimates of numbers and proportions of HIV-1 recombinants in each time period. The systematic review is registered with PROSPERO, CRD42017067164.Findings: Our global data collection yielded an HIV-1 molecular epidemiology database of 383 519 samples from 116 countries in 1990-2015. We found that the proportion of recombinants increased over time, both globally and in most regions, reaching 22·8% (7 978 517 of 34 921 639) of global HIV-1 infections in 2010-15. Both the proportion and the number of distinct CRFs detected increased over time to 16·7% and 57 CRFs in 2010-15. The global and regional distribution of HIV-1 recombinants was diverse and evolved over time, and we found large regional variation in the numbers (0-44 CRFs), types (58 distinct CRFs), and proportions (0-80·5%) of HIV-1 recombinants. Globally, CRF02_AG was the most prevalent recombinant, accounting for 33·9% (2 701 364 of 7 978 517) of all recombinant infections in 2010-15. URFs accounted for 26·7% (2 131 450 of 7 978 517), CRF01_AE for 23·0% (1 838 433), and other CRFs for 16·4% (1 307 270) of all recombinant infections in 2010-15. Although other CRFs accounted for small proportions of infections globally (

Summary Background The 2018 Ebola virus disease (EVD) outbreak in Equateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). Methods We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Equateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. Findings A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name “Tumba”. This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10−3 substitutions per site per year with “Tumba” vs 1·06 × 10−3 substitutions per site per year without “Tumba”). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. Interpretation Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. Funding Defense Biological Product Assurance Office .

Background First ambitious target by 2020 of UNAIDS is that 90% of people living with HIV know their HIV status. In people older than 18 months of age, serological confirmation test is recommended to confirm HIV infection. Case presentation Here we report the case of a patient tested positive with HIV-1, ELISA, Murex® Ag⁄Ab Combination assay (OD450 = 0.802 and cutoff-OD = 0.279) and negative by using FIRST RESPONSE HIV1-2.O CARD TEST (version 2.0) RAPID HIV CARD TEST. Viral load performed with Cobas® TaqMan® 96/Cobas® Ampliprep® was 6.49log10. The virus could be sequenced in partial gag and pol genes and belonged to CRF02_AG clade. Conclusion Conventional PCR is a complementary method for the diagnosis of inconclusive HIV-1 serologies by antibodies.

Global HIV-1 genetic diversity forms a major obstacle to the development of an HIV vaccine. It may be necessary to employ subtype-specific HIV-1 vaccines in individual countries according to their HIV-1 subtype distribution. We estimated the global and regional need for subtype-specific HIV-1 vaccines. We took into account the proportions of different HIV-1 variants circulating in each country, the genetic composition of HIV-1 recombinants, and the different genome segments (gag, pol, env) that may be incorporated into vaccines. We modeled different scenarios according to whether countries would employ subtype-specific HIV-1 vaccines against (1) the most common subtype; (2) subtypes contributing more than 5% of HIV infections; or (3) all circulating subtypes. For therapeutic vaccines targeting the most common HIV-1 subtype in each country, 16.5 million doses of subtype C vaccine were estimated globally, followed by subtypes A (14.3 million) and B (4.2 million). A vaccine based on env required 2.6 million subtype E doses, and a vaccine based on pol required 4.8 million subtype G doses. For prophylactic vaccines targeting the most common HIV-1 subtype in each country, 1.9 billion doses of subtype A vaccine were estimated globally, followed by subtype C (1.1 billion) and subtype B (1.0 billion). A vaccine based on env required 1.2 billion subtype E doses, and a vaccine based on pol required 0.3 billion subtype G doses. If subtype-specific HIV-1 vaccines are also directed against less common subtypes in each country, vaccines targeting subtypes D, F, H, and K are also needed and would require up to five times more vaccine doses in total. We conclude that to provide global coverage, subtype-specific HIV-1 vaccines need to be directed against subtypes A, B, and C. Vaccines targeting env also need to include subtype E and those targeting pol need to include subtype G.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak—between 14 February and 19 June 2021—near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization. The viral lineage responsible for the February 2021 outbreak of Ebola virus disease in Guinea is nested within a clade that predominantly consists of genomes sampled during the 2013–2016 epidemic, suggesting that the virus might have re-emerged after a long period of latency within a previously infected individual.

Objective Seventeen years after the start of the IBAARV (Beninese initiative for access to antiretrovirals), transmitted drug resistance mutations in ARV naïve patients and HIV-1 genetic diversity were investigated in Benin.Results Drug resistance mutations were detected in (27/248; 10.9%) according to the WHO SDRM 2009 list, with a predominance of mutations directed to NNRTIs drugs (24/248; 10%). Phylogenetic and recombination analyses showed a predominance of CRF02_AG strains (165/248; 66.5%) and a high genetic diversity with five other variants and 39 URFs (15.7%) which contained portions of strains that co-circulate in Benin. Eight recent transmission chains revealed active ongoing transmission of HIV-1 strains among ARV naïve patients. Our study showed a moderate primary drug resistance mutations rate and also provided recent data on the HIV-1 variants that circulate in Benin. Regular monitoring of primary drug resistance is required to adapt HIV-1 treatment strategies and adoption of new WHO recommendations in Benin.

BackgroundSeventeen years after the start of the IBAARV (Beninese initiative for access to antiretrovirals), transmitted drug resistance mutations in ARV naïve patients and HIV-1 genetic diversity were investigated in Benin.Methods353 plasma samples were collected between October and December 2017 in nineteen facilities care in Benin from HIV-1 positive and ARV naive individuals. Pol (protease + partial RT) region was amplified and sequenced in 248 samples.ResultsDrug resistance mutations were detected in (27/248; 10.9%) according to the WHO SDRM 2009 list, with predominance of mutations directed to NNRTIs drugs (24/248; 10%).Phylogenetic and recombination analyses showed a predominance of CRF02_AG strains (165/248; 66.5%) and a high genetic diversity with five other variants and 39 URFs (15.7%) which contained portions of strains that co-circulate in Benin. Eight recent transmission chains revealed active ongoing transmission of HIV-1 strains among ARV naïve patients.ConclusionsOur study showed a high primary drug resistance rate and a complex genetic diversity. Regular monitoring of primary drug resistance is required to adapt HIV-1 treatment strategies and adoption of new WHO recommendations in Benin.

Since the ignition of the HIV-1 group M pandemic in the beginning of the 20th century, group M lineages have spread heterogeneously throughout the world. Subtype C spread rapidly through sub-Saharan Africa and is currently the dominant HIV lineage worldwide. Yet the epidemiological and evolutionary circumstances that contributed to its epidemiological expansion remain poorly understood. Here, we analyse 346 novel pol sequences from the DRC to compare the evolutionary dynamics of the main HIV-1 lineages, subtypes A1, C and D. Our results place the origins of subtype C in the 1950s in Mbuji-Mayi, the mining city of southern DRC, while subtypes A1 and D emerged in the capital city of Kinshasa, and subtypes H and J in the less accessible port city of Matadi. Following a 15-year period of local transmission in southern DRC, we find that subtype C spread at least three-fold faster than other subtypes circulating in Central and East Africa. In conclusion, our results shed light on the origins of HIV-1 main lineages and suggest that socio-historical rather than evolutionary factors may have determined the epidemiological fate of subtype C in sub-Saharan Africa., SCOPUS: ar.j, info:eu-repo/semantics/published

Phylogenetic studies have contributed to our understanding of the early epidemic onset of HIV-1 in the Democratic Republic of Congo (DRC); however, the factors driving its early emergence and establishment in human populations still remain unresolved. In order to determine the key aspects of its successful epidemic spread, complete genome data are required from samples representative of the viral diversity in the DRC. In this study, we have established a universal PCR-assay that uses seven different panels of primers to produce overlapping amplicons covering the complete HIV genome. To circumvent the limitations of purifying these fragments and sequencing them with traditional approaches, we have developed a massive parallel sequencing method and a protocol for efficiently assembling HIV-1 genomes. A total of thirty-six samples, collected between 1997 and 2001 from different locations across the DRC, have been obtained, and, at this stage, we are focusing on complementing our dataset with more archival samples that can be used as HIV ‘molecular fossils’. By generating complete genome phylogeographic data from the DRC, we aim to create a genomic window into the past evolutionary and epidemiological dynamics of HIV-1 in Central Africa and understand the natural history of this devastating pandemic.

Summary Background The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. Methods We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. Findings The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Equateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. Interpretation The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. Funding Defense Biological Product Assurance Office.

International audience; Understanding the HIV epidemic in key populations is important. Today only scarce information is available on HIV-1 strains that circulate in men having sex with men (MSM) in sub-Saharan Africa. Here, we studied for the first time the genetic diversity of HIV-1 strains circulating in the MSM population in Lomé, the capital city from Togo. The overall subtype/CRF distribution in pol (protease and/or partial reverse transcriptase (RT)) among the 79 HIV-1 strains from MSM was as follows: CRF02_AG (72%, n=57), subtype G (2.5%, n=2), sub-subtype A3 (1.3%, n=1), and unique recombinant forms (URF) (24%, n=19). Among the 19 URFs four different mosaic structures were observed, annotated as URF1 to URF4. Fifteen sequences (URF1) had the same mosaic structure in pol (G/CRF02_AG) and could represent a new circulating recombinant form (CRF). Phylogenetic analysis of the RT sequences showed that there were several introductions of CRF02_AG strains in the MSM population, however half of the CRF02_AG and all URF1 strains formed a separate, well-supported cluster suggesting one major introduction of CRF02_AG in the MSM population followed by efficient transmission and emergence of a possible new CRF. At least 40% of the strains fell into recent transmission chains involving two to seven MSM. Comparison with >950 HIV-1 sequences from previous studies in Togo showed intermixing of the HIV-1 epidemics between MSM and the general population. Moreover, an HIV-1 strain from a recently HIV-1 infected male patient from Germany, fell within a cluster of HIV-1 strains from MSM from Togo, illustrating recent exchange between MSM from Africa and people from other geographic regions. With growing evidence of the importance of MSM in the dynamic of the HIV epidemic in Africa there is an urgent need for appropriate interventions to limit HIV transmission in this population group.

International audience; Ten days after the declaration of the Ebola outbreak in the Democratic Republic of Congo, rapid identification of the species Zaire Ebola virus using partial gene amplification and nanopore sequencing backed up the use of the recombinant vesicular stomatitis virus-Zaire Ebola virus vaccine in the recommended ring vaccination strategy.

Molecular epidemiological studies revealed that the epicenter of the HIV pandemic was Kinshasa, the capital city of the Democratic Republic of the Congo (DRC) in Central Africa. All known subtypes and numerous complex recombinant strains co-circulate in the DRC. Moreover, high intra-subtype diversity has been also documented. During two previous surveys on HIV-1 antiretroviral drug resistance in the DRC, we identified two divergent subtype C lineages in the protease and partial reverse transcriptase gene regions. We sequenced eight near full-length genomes and classified them using bootscanning and likelihood-based phylogenetic analyses. Four strains are more closely related to subtype C although within the range of inter sub-subtype distances. However, these strains also have small unclassified fragments and thus were named CRF92_C2U. Another strain is a unique recombinant of CRF92_C2U with an additional small unclassified fragment and a small divergent subtype A fragment. The three remaining strains represent a complex mosaic named CRF93_cpx. CRF93_cpx have two fragments of divergent subtype C sequences, which are not conventional subtype C nor the above described C2, and multiple divergent subtype A-like fragments. We then inferred the time-scaled evolutionary history of subtype C following a Bayesian approach and a partitioned analysis using major genomic regions. CRF92_C2U and CRF93_cpx had the most recent common ancestor with conventional subtype C around 1932 and 1928, respectively. A Bayesian demographic reconstruction corroborated that the subtype C transition to a faster phase of exponential growth occurred during the 1950s. Our analysis showed considerable differences between the newly discovered early-divergent strains and the conventional subtype C and therefore suggested that this virus has been diverging in humans for several decades before the HIV/M diversity boom in the 1950s.

The objective of this study was to investigate the performance of the ViroSeq HIV-1 Genotyping System v2.0 on HIV-1 non-B strains identified in Senegalese patients. The study involved 150 patients, and genotyping was performed using the ViroSeq HIV-1 Genotyping System v2.0 or an in-house method developed by the French National Agency on AIDS Research AC11 when the ViroSeq HIV-1 Genotyping System v2.0 failed. The sequences were edited to assess the performance of sequencing primers at their presumed binding regions. The Polymorphism was studied in the regions between the sequences of Senegalese patients and the subtype B strains used as references. The phylogenetic analysis showed a predominance of CRF02_AG (88/150; 58.7%) and the circulation of 11 subtypes/CRFs, 16 unique recombinant forms (URFs) and one unclassified sample. The amplification and sequencing rates were 98% (147/150) and 96.6% (142/147), respectively. This study showed that only primer B exhibited 100% success, while the failure rate ranged from 1.4% to 71.4% for the other primers (D: 71.4%, A and H: 12.2%, F: 7.5%, G: 5.5% and C: 1.4%). These findings suggest the need for an alternative method or alternative primers for non-B strains that were not sequenced successfully using the ViroSeq HIV-1 Genotyping System v2.0.

International audience; With the increasing demand of HIV viral load (VL) tests in resource-limited countries (RLCs) there is a need for assays at affordable cost and able to quantify all known HIV-1 variants. VLs obtained with a recently developed open and polyvalent universal HIV-1/SIVcpz/SIVgor RT-qPCR were compared to Abbott RealTime HIV-1 assay in Cameroon. On 474 plasma samples, characterized by a wide range of VLs and a broad HIV-1 group M genetic diversity, 97.5% concordance was observed when using the lower detection limit of each assay. When using the threshold of 3.00 log10 copies/mL, according to WHO guidelines to define virological failure (VF) in RLCs, the concordance was 94.7%, 360/474 versus 339/474 patients were identified with VF with the new assay and Abbott RealTime HIV-1, respectively. Higher VLs were measured with the new assay, +0.47 log10 copies/mL (95% CI; 0.42-0.52) as shown with Bland-Altman analysis. Eleven samples from patients on VF with drug resistance were not detected by Abbott RealTime HIV-1 versus two only with the new assay. Overall, our study showed that the new assay can be easily implemented in a laboratory in RLCs with VL experience and showed good performance on a wide diversity of HIV-1 group M variants.

In this study, we investigated the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations and genetic variability among Senegalese patients undergoing highly active antiretroviral therapy (ART) in the public health system. We conducted a cross-sectional study of 72 patients with suspected therapeutic failure. HIV-1 genotyping was performed with Viroseq HIV-1 Genotyping System v2.0 or the procedure developed by the ANRS AC11 resistance study group, and a phylogenetic analysis was performed. The median follow-up visit was at 40 (range, 12 to 123) months, and the median viral load was 4.67 (range, 3.13 to 6.94) log 10 copies/ml. The first-line therapeutic regimen was nucleoside reverse transcriptase inhibitors (NRTIs) plus efavirenz (EFV) or NRTIs plus nevirapine (NVP) (54/72 patients; 75%), and the second-line therapy was NRTIs plus a protease inhibitor (PI/r) (18/72; 25%). Fifty-five patients (55/72; 76.39%) had at least one drug resistance mutation. The drug resistance rates were 72.22 and 88.89% for the first-line and second-line ARTs, respectively. In NRTI mutations, thymidine analog mutations (TAMs) were found in 50.79% and the M184V mutation was found in 34.92% of the samples. For non-NRTI resistance, we noted a predominance of the K103N mutation (46.27%). For PI/r, several cases of mutations were found with a predominance of M46I and L76V/F at 24% each. The phylogenetic analysis revealed CRF02_AG as the predominant circulating recombinant form (43/72; 59.72%). We found a high prevalence of resistance mutations and a high rate of TAMs among Senegalese patients in the public health system. These findings emphasize the need to improve virological monitoring in resource-limited settings.

In this study, we characterized HIV-1 strains from the Democratic Republic of Congo (DRC), previously described as divergent subtype A (n = 1, 97CD.KMST91) or untypable (n = 7) in the V3–V5 env region. Four strains had the same structure over the entire genome, including alternating fragments of a new subsubtype, A5, within the subtype A radiation and fragments that remain unclassified. Therefore, the cluster of new viruses represents a new circulating recombinant, CRF26_A5U. Three additional strains were unique recombinants with the newly described CRF26_A5U and subtype C. Finally, the nearly full-length sequence of 97CD.KMST91 showed that this strain also consisted of alternating fragments of a divergent subtype A lineage and unclassified fragments, although different from previously reported A and U sequences. The high genetic distances among the different CRF26-A5U strains suggest their longstanding presence in the DRC.

Background In this work, we investigated the genetic diversity of HIV-1 and the presence of mutations conferring antiretroviral drug resistance in 50 drug-naïve infected persons in the Republic of Congo (RoC). Samples were obtained before large-scale access to HAART in 2002 and 2004. Methods To assess the HIV-1 genetic recombination, the sequencing of the pol gene encoding a protease and partial reverse transcriptase was performed and analyzed with updated references, including newly characterized CRFs. The assessment of drug resistance was conducted according to the WHO protocol. Results Among the 50 samples analyzed for the pol gene, 50% were classified as intersubtype recombinants, charring complex structures inside the pol fragment. Five samples could not be classified (noted U). The most prevalent subtypes were G with 10 isolates and D with 11 isolates. One isolate of A, J, H, CRF05, CRF18 and CRF37 were also found. Two samples (4%) harboring the mutations M230L and Y181C associated with the TAMs M41L and T215Y, respectively, were found. Conclusion This first study in the RoC, based on WHO classification, shows that the threshold of transmitted drug resistance before large-scale access to antiretroviral therapy is 4%.

International audience; OBJECTIVE:In resource-limited countries, antiretroviral therapy (ART) has been scaled up, but individual monitoring is still suboptimal. Here, we studied whether or not ART had an impact on the frequency and selection of drug resistance mutations (DRMs) under these settings. We also examined whether differences exist between HIV-1 genetic variants.DESIGN:A total of 3736 sequences from individuals failing standard first-line ART (n = 1599, zidovudine/stavudine + lamivudine + neviparine/efavirenz) were analyzed and compared with sequences from reverse transcriptase inhibitor (RTI)-naive individuals (n = 2137) from 10 West and Central African countries.METHODS:Fisher exact tests and corrections for multiple comparisons were used to assess the significance of associations.RESULTS:All RTI-DRM from the 2015 International Antiviral Society list, except F227C, and nine mutations from other expert lists were observed to confer extensive resistance and cross-resistance. Five additional independently selected mutations (I94L, L109I, V111L, T139R and T165L) were statistically associated with treatment. The proportion of sequences with multiple mutations and the frequency of all thymidine analog mutations, M184V, certain NNRTIS, I94L and L109I showed substantial increase with time on ART. Only one nucleoside and two nonnucleoside RTI-DRMs differed by subtype/circulating recombinant form.CONCLUSION:This study validates the global robustness of the actual DRM repertoire, in particular for circulating recombinant form 02 predominating in West and Central Africa, despite our finding of five additional selected mutations. However, long-term ART without virological monitoring clearly leads to the accumulation of mutations and the emergence of additional variations, which limit drug options for treatment and can be transmitted. Improved monitoring and optimization of ART are necessary for the long-term effectiveness of ART.

International audience; BACKGROUND:Access to antiretroviral treatment (ART) in resource-limited countries has increased significantly but scaling-up ART into semi-rural and rural areas is more recent. Information on treatment outcome in such areas is still very limited notably due to additional difficulties to manage ART in these areas.RESULTS:387 HIV-1 infected adults (≥18 years) were consecutively enrolled when attending healthcare services for their routine medical visit at 12 or 24 months on first-line ART in five HIV care centers (four semi-rural and one rural). Among them, 102 patients were on first-line ART for 12 ± 2 months (M12) and 285 for 24 ± 2 months (M24). Virological failure was observed in 70 (18.1 %) patients ranging from 13.9 to 31.6 % at M12 and from 8.1 to 22.4 % at M24 across the different sites. For 67/70 patients, sequencing was successful and drug resistance mutations were observed in 65 (97 %). The global prevalence of drug resistance in the study population was thus at least 16.8 % (65/387). Moreover, 32 (8.3 %) and 27 (6.9 %) patients were either on a completely ineffective ART regime or with only a single drug active. Several patients accumulated high numbers of mutations and developed also cross-resistance to abacavir, didanosine or the new NNRTI drugs like etravirine and rilpivirine.CONCLUSION:The observations on ART treatment outcome from ART clinics in semi-rural areas are close to previous observations in Lomé, the capital city suggesting that national ART-programme management plays a role in treatment outcome.

The Democratic Republic of Congo (DRC) is characterized by low and stable HIV prevalences and high HIV-1 genetic diversity and is most probably the epicenter of HIV-1 group M. Our major goal was to study the distribution of HIV-1 variants over a 5-year period against a background of political instability and civil war. A total of 288 HIV-1-positive samples collected in 2002 from sentinel population groups in an HIV serosurveillance study performed in 4 cities (Kinshasa [capital city], Mbuji-Mayi [south], Lubumbashi [southeast], and Kisangani [northeast]) were genetically characterized by sequencing and phylogenetic analysis of the V3-V5 env region. The results were compared with those obtained in 1997. Similarly, as in 1997, an extremely high genetic diversity of HIV-1 strains overall and a heterogeneous geographic distribution were seen in 2002. All subtypes and several circulating recombinant forms were present, high intrasubtype diversity was observed, and 5.6% of the samples could not be classified. In each geographic region of the DRC, the genetic diversity was significantly higher than in neighboring countries. Comparison of subtype distribution in similar population groups in Kinshasa in 1997 and 2002 revealed an overall increase of subtype C in Kinshasa from 2.1% to 9.7% and, more precisely, from 0% to 18.9% in female sex workers (P = 0.013). Genetic characterization of HIV-positive samples from sentinel surveys adds significant additional information on new trends in the HIV epidemic. These changes could have implications regarding the spread of HIV infection in the DRC as well on vaccine and/or treatment strategies.

On the basis of partial env and gag subtyping, we documented that the majority of HIV-1 strains circulating in Niger were CRF02-AG (54.3%) or CRF06-cpx (18.1%) and that 9% of the samples were possible recombinants between CRF02 and CRF06. To determine in more detail the precise structure of these viruses we sequenced the full-length genomes for three such strains (97NE-003, 00NE-036, and 00NE-095). From the bootscan and phylogenetic tree analysis it is evident that the new viruses are the result of recombination events between CRF02-AG and CRF06-cpx strains. Importantly, each virus had a different complex recombinant structure with multiple breakpoints, leading to viruses with complex mosaic patterns.

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen., In this individual patient and sequence-level meta-analysis, Soo-Yon Rhee and colleagues measure regional trends in HIV-1 transmitted drug resistance prevalence and investigate the specific mutations responsible for TDR in different regions and in different virus subtypes., Editors' Summary Background About 35 million people are currently infected with HIV, the virus that causes AIDS by destroying immune system cells and leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years of infection. Then, in 1996, effective antiretroviral (ARV) therapy—drug combinations that suppress HIV replication by inhibiting reverse transcriptase and other essential viral enzymes—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ARV therapy was expensive, HIV/AIDS remained fatal in low- and middle-income countries (LMICs). In 2003, the international community began to work towards achieving universal access to ARV therapy. Now, more than 10 million HIV-positive individuals in LMICs receive ARV therapy, usually as a fixed-dose combination of two nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir and lamivudine, plus a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as efavirenz or nevirapine. Why Was This Study Done? The global scale-up of ARV therapy has reduced deaths from HIV/AIDS and the incidence of HIV infection in LMICs, but the development of resistance to ARV therapy is threatening these advances. HIV rapidly accumulates genetic changes (mutations), some of which make HIV resistant to ARV therapy. Up to 30% of patients receiving a fixed-dose NRTI/NNRTI combination develop virological failure, and a high proportion of these patients develop mutations associated with resistance to the ARVs in their regimen. Moreover, the proportion of newly infected, ARV-naïve individuals with transmitted drug resistance (TDR) is also increasing. Organizations involved in HIV/AIDS control need to understand the regional and temporal mutational patterns of TDR to inform the development of guidelines for first-line ARV therapy and of inexpensive resistance mutation assays for use in LMICs. Here, using a statistical approach called meta-analysis to combine information from individual patients about the resistance mutations they carry, the researchers investigate the molecular epidemiology of TDR (the patterns of molecular changes underlying TDR in populations) and identify the HIV drug-resistance mutations most responsible for TDR in different world regions. What Did the Researchers Do and Find? The researchers identified 287 studies published between 2000 and 2013 from 111 countries that included the reverse transcriptase sequences of HIV viruses from 50,870 ARV-naïve, HIV-positive individuals. The researchers analyzed each virus sequence for the presence of 93 surveillance drug-resistance mutations (SDRMs) previously shown to be specific indicators of TDR. Meta-analysis of these data indicated that the average overall prevalence of TDR (the proportion of ARV-naïve, HIV-positive individuals infected with a virus carrying one or more SDRMs) ranged from 2.8% in sub-Saharan Africa to 11.5% in North America. In sub-Saharan Africa, the odds (chance) of TDR increased 1.09-fold per year following national ARV scale-up; this increase was attributable to an increase in NRTI- and NNRTI-associated resistance. By contrast, in LMICs in south/southeast Asia, the odds of TDR remained unchanged following ARV scale-up. In Latin America/Caribbean, North America, Europe, and upper-income Asian countries, the odds of TDR have increased by around 1.10-fold per year since 1995, mainly as a result of increased NNRTI resistance. Four NNRTI-associated and 16 NRTI-associated SDRMs accounted for most NNRTI- and NRTI-associated TDR, respectively, in all regions. Notably, in sub-Saharan Africa and south/southeast Asia, most of the NNRTI-associated SDRMs detected were associated with high-level resistance to nevirapine or efavirenz. Finally, the researchers report that 95% of TDR viruses in sub-Saharan Africa and south/southeast Asia were unrelated and had therefore arisen independently. What Do These Findings Mean? Because many drug-resistance mutations reduce HIV’s fitness and tend to be lost rapidly in individuals not exposed to ARV therapy, differences among the datasets used in this meta-analysis with respect to how long each ARV-naïve patient had been infected with HIV before virus sampling may limit the accuracy of these findings. Nevertheless, the finding that most of the TDR strains detected in sub-Saharan Africa and south/southeast Asia arose independently suggests that improved patient adherence to ARV therapy and the use of ARV regimens that contain drugs to which HIV rarely develops resistance (regimens with a high genetic barrier to resistance) should reduce the generation of new ARV-resistant strains and mitigate TDR increases. In addition, the finding that a few NNRTI-resistance mutations were responsible for most cases of transmitted high-level resistance suggests that an inexpensive assay that detects these specific mutations may be useful for pre-therapy screening in LMICs with high TDR levels. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001810. Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, and personal stories about living with HIV/AIDS Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on antiretroviral drugs and on universal access to ARV therapy; Avert also provides personal stories about living with HIV/AIDS The World Health Organization provides information on all aspects of HIV/AIDS (in several languages), including its guidelines on the use of antiretroviral therapy for treating and preventing HIV infection The UNAIDS World AIDS Day Report 2014 provides up-to-date information about the AIDS epidemic and efforts to halt it, including progress towards universal access to antiretroviral therapy The Stanford University HIV Drug Resistance Database includes information about surveillance drug-resistant mutations (SDRMs) and an interactive map displaying HIV drug resistance in ARV-naïve populations

Up to now, all known env subtype E viruses (CRF01-AE) have had the same mosaic structure with subtype A, and no other env subtype E HIV-1 viruses with non-A subtypes in their genomes have been described. In this report we describe the full-length genome sequence of an env subtype E isolate with a recombinant genome different from the prototype CRF01-AE strains. The 97CD-KTB49 strain, obtained from a tuberculosis patient in Kinshasa, has a complex mosaic genome involving subtypes A, E, G, H, J, K, and several unknown fragments. The U sequences formed well-separated clusters together with previously described unknown fragments from CRF04-cpx (subtype I), and from Z321, the oldest intersubtype recombinant isolated in 1976 in the Democratic Republic of Congo. The complex recombinant virus from our study is not an isolated strain; partial sequencing of a second strain, 97CD-KFE45, confirmed the breakpoints observed in the 97CD-KTB49 strain in the regions sequenced. The complexity of these recombinant strains suggests a longstanding presence of subtype E in Central Africa.

Background: Treatment of cytomegalovirus (CMV) diseases with protracted administration of ganciclovir can promote the development of resistant CMV that is associated with a poor response to therapy. It has been shown that the majority of ganciclovir-resistant CMV isolates carry mutations in the UL97 phosphotransferase gene. Objectives: To evaluate the frequency of CMV resistance to ganciclovir in patients with AIDS treated with ganciclovir and to identify the UL97 gene mutations associated with ganciclovir resistance. Study design: Analysis of CMV blood isolates obtained over 1 year from patients treated with ganciclovir. CMV susceptibility to ganciclovir was determined by an immediate early antigen plaque reduction assay; UL97 gene mutations were identified by restriction enzyme digest analysis and sequencing. Results: Twenty-nine patients were followed-up; 17 CMV blood isolates were obtained from 10 ganciclovir-experienced patients. Thirteen (76%) of these isolates, obtained from seven (24%) patients after a median treatment duration of 5.5 months, were resistant to ganciclovir. Five of the seven patients who had a ganciclovir-resistant CMV in blood showed retinitis progression. UL97 gene mutations were identified in nine CMV isolates at codons 460 (M → V), 594 (A → V and A → T), and 595(L → S). Three patients developed a ganciclovir-resistant virus after a ganciclovir treatment shorter than 60 days (28–58 days). In another patient, we observed that ganciclovir resistance persisted 4 months after discontinuation of ganciclovir therapy. Conclusion: Our results indicate that ganciclovir resistance due to UL97 gene mutations is common in subjects with AIDS-related CMV diseases treated with ganciclovir. Detection of these mutations represents a tool for the management of patients with ganciclovir therapy.

Circulating and unique recombinant HIV-1 strains continue to be identified and their number increases over time, suggesting that co-infection with multiple HIV-1 is frequent. In this study we analyzed to what extent dual infections with different HIV-1 variants occur in a population group with high risk behaviour, high HIV-1 prevalence and in an area where multiple HIV-1 subtypes and Circulating Recombinant Forms (CRFs) co-circulate. We studied 69 MSM with our recently developed multi-region hybridization assay (MHA), based on fluorescent probe detection for eight common variants circulating in West and West Central Africa. At least 11 (15.9%) of the 69 patients were simultaneously infected with two different HIV-1 subtypes and/or CRFs. Among the 29 samples identified as subtype C by MHA in gag, 15 (57.7%) reacted with both C1 and C2 probes. Sequence analysis suggests that the majority of the samples reactive with C1 and C2 probes are most likely infected with two different subtype C clades. Single genome amplification and DNA dilutions confirmed dual infection with subtype D and C for MSM1193, triple infection with two different C subtype strains and one CRF02_AG strain in MSM1157 and showed that MSM3017 is at least co-infected with CRF06_cpx and CRF02_AG and another strain that could not be classified. Comparison of all subtype C sequences from the MSM population and from the general population from this and previous studies confirmed the intermixing of HIV-1 variants between low-risk women and high-risk men as shown by the intermixing of subtype C variants from MSM1157 and a female patient (02SN-HALD478). Comparison of dual infection rates between the general population and MSM in Senegal, show also clearly the importance of high HIV prevalence and high risk behavior in dual infections and subsequent intermixing of HIV-1 variants which can lead to emergence and spread of new recombinants (CRFs).

HIV-1 epidemics are expanding among men who have sex with men in low- and middle-income countries. To confirm and further explore preliminary data in Senegal, we aimed to determine 3 years after a first study the HIV-1 genetic diversity in three different viral regions. From 109 samples available in 2007, 93 were sequenced in gag, 77 in env, and 60 in pol. Phylogenetic analysis showed that subtype C predominated (38–52%), followed by CRF02_AG (30–40%), subtype B (13–17%), and CRF09_cpx (2.6–5%). Subsubtype A3 and strains tightly linked to CRF43_02G were identified in env and gag, respectively, and 12% of the samples were unique recombinants. Six transmission chains involving two to seven individuals were identified. Some strains carried resistance mutations inside transmission chains. This study confirmed the existence of a dual epidemic in Senegal and emphasized the need to strengthen prevention programs to avoid strains intermixing between low-risk women and high-risk men.

In West and West Central Africa, multiple subtypes, circulating recombinant forms (CRF), and high proportions of unique recombinant forms (URF) are documented. The predominance of recombinants strongly suggests that dual infections occur frequently. In the present study, we adapted the multi-region hybridization assay (MHA), previously developed to identify dual infections in geographic regions where few HIV-1 variants circulate, to identify HIV-1 variants and dual infections. We designed clade-specific probes in three genomic regions ( gag p17, vpu , nef ) to detect eight different variants that are common in this part of Africa (A, B/D, C, F, G, CRF02_AG, CRF06_cpx, CRF22_01A1). The assay was validated with 163 samples representing the corresponding HIV-1 variants. Depending on the genomic regions, the global sensitivity of the assay ranged from 86% to 94%, and the global specificity was between 85% and 96%. The assay was then applied on 156 antiretroviral treatment-naive patients from Cameroon. The MHA assay identified 79%, 85% and 90% of the strains in nef , gag and vpu regions, respectively. The subtype/CRF distribution and the proportion of inter-region recombinants obtained by the new MHA assay were in accordance with known subtype/CRF distribution in Cameroon. Moreover, the MHA assay identified 35 (22.4%) patients as dually infected, from which 20 were reactive in more than one region and/or with concordant multigenomic recombination pattern. Despite the high genetic diversity, we successfully developed an hybridization assay allowing identification of eight common HIV-1 variants circulating in West and West Central Africa. We documented high rates of dual infection in a low-risk population group, illustrating that the global evolution of HIV diversity is driven by dual infections. This assay could become a useful screening tool for the global surveillance and monitoring of inter-subtype/CRF dual infections in West and West Central Africa.

Background Although the outbreak of human immunodeficiency virus type 1 (HIV-1) in Guangdong has been documented for more than a decade, the molecular characteristics of such a regional HIV-1 epidemic remained unknown. Methodology/Principal Findings By sequencing of HIV-1 pol/env genes and phylogenetic analysis, we performed a molecular epidemiologic study in a representative subset (n = 200) of the 508 HIV-1-seropositive individuals followed up at the center for HIV/AIDS care and treatment of Guangzhou Hospital of Infectious Diseases. Of 157 samples (54.1% heterosexual acquired adults, 20.4% needle-sharing drug users, 5.7% receivers of blood transfusion, 1.3% men who have sex with men, and 18.5% remained unknown) with successful sequencing for both pol and env genes, 105 (66.9%) HIV-1 subtype CRF01_AE and 24 (15.3%) CRF07_BC, 9 (5.7%) B’, 5 (3.2%) CRF08_BC, 5 (3.2%) B, 1 (0.6%) C, 3 (1.9%) CRF02_AG, and 5 (3.2%) inter-region recombinants were identified within pol/env sequences. Thirteen (8.3%) samples (3 naives, 6 and 5 received with antiretroviral treatment [ART] 1–21 weeks and ≥24 weeks respectively) showed mutations conferring resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors or protease inhibitors. Among 63 ART-naive patients, 3 (4.8%) showed single or multiple drug resistant mutations. Phylogenetic analysis showed 8 small clusters (2–3 sequences/cluster) with only 17 (10.8%) sequences involved. Conclusion/Significance This study confirms that sexual transmission with dominant CRF01_AE strain is a major risk for current HIV-1 outbreak in the Guangdong’s general population. The transmission with drug-resistant variants is starting to emerge in this region.

International audience; Background: The classification of HIV-1 strains in subtypes and Circulating Recombinant Forms (CRFs) has helped in tracking the course of the HIV pandemic. In Senegal, which is located at the tip of West Africa, CRF02_AG predominates in the general population and Female Sex Workers (FSWs). In contrast, 40% of Men having Sex with Men (MSM) in Senegal are infected with subtype C. In this study we analyzed the geographical origins and introduction dates of HIV-1 C in Senegal in order to better understand the evolutionary history of this subtype, which predominates today in the MSM population Methodology/Principal Findings: We used a combination of phylogenetic analyses and a Bayesian coalescent-based approach, to study the phylogenetic relationships in pol of 56 subtype C isolates from Senegal with 3025 subtype C strains that were sampled worldwide. Our analysis shows a significantly well supported cluster which contains all subtype C strains that circulate among MSM in Senegal. The MSM cluster and other strains from Senegal are widely dispersed among the different subclusters of African HIV-1 C strains, suggesting multiple introductions of subtype C in Senegal from many different southern and east African countries. More detailed analyses show that HIV-1 C strains from MSM, are more closely related to those from southern Africa. The estimated date of the MRCA of subtype C in the MSM population in Senegal is estimated to be in the early 80's. Conclusions/Significance: Our evolutionary reconstructions suggest that multiple subtype C viruses with a common ancestor originating in the early 1970s entered Senegal. There was only one efficient spread in the MSM population, which most likely resulted from a single introduction, underlining the importance of high risk behavior in spread of viruses.

Background: We studied virological outcome and drug resistance in patients on antiretroviral therapy (ART) in health care centers in the Democratic Republic of Congo and looked for the presence of drug resistance in antiretroviral-naive patients attending the same clinics. Methods: In 2008, we conducted a cross-sectional survey among patients on ART for >= 12 months in 4 major cities [Kinshasa (n = 289), Matadi (n = 198), Lubumbashi (n = 77), and Mbuji-Mayi (n = 103)]. Genotypic drug resistance tests were done with an in-house assay on samples with viral load > 1000 copies/mL. ART-naive patients (n = 283) were also consecutively enrolled in the same clinics. Results: Of the 667 patients on ART, > 98% received Lamivudine + Stavudine/azidothymidine + Nevirapine/Efavirenz as first-line regimen and 74.4% were women. Median time on ART was 25 months [interquartile ratio (IQR), 19-32] in Kinshasa, 26 months (IQR, 19-32) in Matadi, 27 months (IQR, 19-44) in Lubumbashi, and 19 months (IQR, 16-24) in Mbuji-Mayi. A total of 97 patients (14.6%) had viral load > 1000 copies/mL, and among the 93 successfully sequenced samples, 78 (83.9%) were resistant to at least 1 drug of their ART regimen: 68 harbored resistance mutations to nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI), 2 to NRTI only, 7 to NNRTI only, and 1 to NRTI + NNRTI + protease inhibitor. The majority of patients, 70/78 (89.7%), were resistant to at least 2 of the 3 drugs from their treatment. The use of next-generation NNRTI, etravirine was already compromised for 19.2% (15/78) of the patients and 7 patients had the K65R mutation compromising the use of tenofovir in second-line regimens. The proportion of antiretroviral-resistant patients increased over time from 8.4% to 18.6% for patients on ART for 12-23 months or > 35 months (P = 0.013), respectively. Virological failure and rates of drug resistance were significantly higher among men than women, 19.9% versus 8.8%, respectively (P = 0.0001). Among the 253 recently diagnosed patients, 20 (7.9%) harbored resistance mutations. Conclusions: The accumulation of drug resistance mutations with time on ART needs further attention, and surveillance should be reinforced in ART programs in sub-Saharan Africa.

For the first time the genetic diversity among the uniformed personnel in Kinshasa, the capital city of the Democratic Republic of Congo (DRC), a country that has experienced military conflicts since 1998 and in which the global HIV-1/M pandemic started, has now been documented. A total of 94 HIV-1-positive samples, collected in 2007 in Kinshasa garrison settings from informed consenting volunteers, were genetically characterized in the pol region (protease and RT). An extensive diversity was observed, with 51% of the strains corresponding to six pure subtypes (A 23%, C 13.8%, D, G, H, J, and untypable), 15% corresponding to nine different CRFs (01, 02, 11, 13, 25, 26, 37, 43, and 45), and 34% being unique recombinants with one-third being complex mosaic viruses involving three or more different subtypes/CRFs. Only one strain harbored a single mutation, I54V, associated with drug resistance to protease inhibitors. Due to their high mobility and potential risk behavior, HIV infections in military personnel can lead to an even more complex epidemic in the DRC and to a possible increase of subtype C.

In this study, HIV strains circulating among military personnel were characterized, in Malabo, the capital city of Equatorial Guinea. One sample was found to be HIV-2 group A while a high degree of genetic diversity was recorded in the pol region of 41 HIV-1-positive samples. CRF02_AG accounted for 53.7% of the strains, and 11 different variants were obtained in the remaining 19 samples: subtype G (n = 3), A3 (n = 2), C (n = 2), CRF26_A5U (n = 2), F2 (n = 1), CRF06 (n = 1), CRF09 (n = 1), CRF11 (n = 1), CRF22 (n = 1), and divergent subtype A (n = 1) and F (n = 1). One strain could not be classified and three were unique recombinants. Analysis of antiretroviral drug resistance mutations revealed two patients each harboring one major mutation, M46I in protease and D67N in reverse transcriptase sequences, respectively. The high genetic diversity and emerging ARV resistance mutations call for frequent surveys and appropriate monitoring of ARV considering the increasing access to ARV in the country.

BACKGROUND: Recent reports showed the high vulnerability for HIV infection of men who have sex with men (MSM) in Africa. Here, we report the HIV-1 variants that circulate among MSM in Senegal. METHODS: HIV-1 subtype/circulating recombinant form (CRF) was determined in an 1800-base pair fragment of pol for 70 HIV-1-positive samples from MSM. Phylogenetic trees were constructed using the neighbor-joining method with CLUSTALX. Similarity and bootstrap plots were then done for recombination analysis. The maximum likelihood approach was used for the identification of transmission clusters. RESULTS: Sixty-seven samples (95%) were from Senegalese MSM, 90% unmarried with a median age of 30 years. Fifty-five MSM had regular male partners, but 39 of 70 had also a regular female partner. The overall subtype/CRF distribution was as follows: 28 C (40%), 17 CRF02_AG (24.3%), 13 B (18.6%), 6 G (8.6%), 3 CRF09_cpx (4.3%), and 3 (4.3%) unique recombinants. In addition, 47 sequences (67.15%) were segregated into 15 transmission clusters. CONCLUSIONS: These variants circulate also among the general population or female sex workers, but the proportions are significantly different. Despite the massive stigma, the majority (80%) of MSM recognized having sex with women and could serve as a bridge for intermixing of HIV-1 variants between high-risk men and low-risk women.

Full-length genomes were characterized for two samples, 02CD-LBR024 from the Democratic Republic of Congo (DRC) and 04FR-CD-KZS from France, that formed a separate subcluster with a previously characterized env subtype E isolate from DRC with a recombinant structure different from CRF01-AE. Since the three viruses are clearly epidemiologically unlinked and share the same complex recombinant structure, they represent a circulating recombinant form, designated as CRF27-cpx. The recombination pattern involves six different HIV-1 subtypes (A, E, G, H, J, and K) and a small unclassified fragment. The genetic distances are relatively high, indicating that CRF27-cpx evolved over a long time. Their prevalence is low (0.75%) and remained stable over time in the DRC. The existence of the 04FR. CD. KZS virus, in a patient who recently seroconverted in France, confirmed that these strains now circulate outside the DRC. Continuous monitoring of HIV-1 strains thus remains important to allow early identification of the introduction of new variants.

Since the first outbreak of HIV-1 was reported in heroin users in China in 1989, HIV-1 has spread steadily among injection drug users, leading to an exponential growth of nationwide outbreaks from 1998 to 2004. However, the impact of sexual transmission on outbreaks of HIV in China's general population is still unclear. Through a governmental HIV/AIDS surveillance program, an HIV serological study was conducted in volunteers between 1996 and 2005 in Xishuangbanna Dai Autonomous Prefecture of Yunnan province. We performed the transmission reconstruction by molecular epidemiological tracing in a subset of the HIV-1-seropositive individuals diagnosed during this survey. Neighbor joining and maximum likelihood trees based on the HIV-1 pol and env genes were implemented to provide information on putative epidemiological links, which were then confirmed by contact tracing. Of 25,390 volunteers, 501 (2%) accumulated cases of HIV-1 infection (21.1% in needle-sharing drug users, 77.3% in heterosexual adults, 0.4% in homosexual adults, and 1.2% in children born from infected mothers) were diagnosed. Among 44 heterosexually infected and antiretroviral-naive local-traceable individuals (27 infected with HIV-1 subtype CRF01_AE, 15 with CRF08_BC, 1 with G, and 1 with a new B/C recombinant), 18 (40.9%) were coclustered into 8 transmission chains with an average size of 2.25 infections per chain. Phylogenetic and epidemiological linkages confirmed eight heterosexual transmission events. This is the first report providing molecular epidemiological evidence of heterosexual transmission of HIV-1 in China's general population. The reconstruction of transmission of current HIV-1 outbreaks by molecular epidemiological tracing is instrumental in identifying sources of the epidemic and in defining prevention strategies.

Human immunodeficiency virus type 1 (HIV-1) is classified in nine subtypes (A to D, F, G, H, J, and K), a number of subsubtypes, and several circulating recombinant forms (CRFs). Due to the high level of genetic diversity within HIV-1 and to its worldwide distribution, this classification system is widely used in fields as diverse as vaccine development, evolution, epidemiology, viral fitness, and drug resistance. Here, we demonstrate how the high recombination rates of HIV-1 may confound the study of its evolutionary history and classification. Our data show that subtype G, currently classified as a pure subtype, has in fact a recombinant history, having evolved following recombination between subtypes A and J and a putative subtype G parent. In addition, we find no evidence for recombination within one of the lineages currently classified as a CRF, CRF02_AG. Our analysis indicates that CRF02_AG was the parent of the recombinant subtype G, rather than the two having the opposite evolutionary relationship, as is currently proposed. Our results imply that the current classification of HIV-1 subtypes and CRFs is an artifact of sampling history, rather than reflecting the evolutionary history of the virus. We suggest a reanalysis of all pure subtypes and CRFs in order to better understand how high rates of recombination have influenced HIV-1 evolutionary history.

In 2002, an HIV surveillance study was performed among more than 5500 individuals representing the general population of urban and rural districts in Burundi. In this report, we genetically characterized a subset of the HIV-1-positive samples identified during this survey, including all the HIV-positive samples from Bujumbura, the capital city, and samples from one semiurban and one rural district. One hundred and nineteen samples were genetically characterized in the V3-V5 region of the env gene and/or in the protease and reverse transcriptase region of the pol gene. Phylogenetic analysis of 101 env/pol sequences revealed that the HIV-1 epidemic in Burundi was driven by subtype C (81.2%), followed by subtype A (7.9 %) and polC/envA recombinants (5.9%). One major mutation associated with resistance to antiretroviral drugs (ARVs) in the pol gene, as defined by the International AIDS Society Resistance Testing-USA panel, was observed in one individual, but many minor resistance-associated mutations were also present in the majority of the samples.

A molecular and phylogenetic characterization on env and gag subgenomic regions has been performed in our laboratory on HIV-1 variants identified in seropositive individuals residing in Italy, infected in the 1999-2001 period, and five non-B-subtype HIV-1 isolates have been described. To confirm the phylogenetic characterization and to determine the genomic organization of three non-B HIV-1 isolates (A, G, and CRF02- AG), the complete gag, pol, and gp120 ORFs (approx. 6900 bp) have been sequenced for each of them. The phylogenetic tree analyses performed on the whole sequence or on individual genes suggested, for the A and G isolates, the identification of divergent strains that do not cluster into any of the known subsubtypes. This has been further validated by pairwise distance analysis. On the contrary, the phylogenetic classification of the CRF02-AG isolate has been confirmed and an overall typical pattern of intragenomic breakpoints has been observed by a Simplot analysis. These results confirm the constant HIV-1 molecular evolution and indicate the relevance of a continuous molecular monitoring of HIV-1 isolates for the development of appropriate vaccine candidates.

Background CRF02_AG is the predominant HIV strain circulating in West and West Central Africa. The aim of this study was to test whether this predominance is associated with a higher in vitro replicative fitness relative to parental subtype A and G viruses. Primary HIV-1 isolates (10 CRF02_AG, 5 subtype A and 5 subtype G) were obtained from a well-described Cameroonian cohort. Growth competition experiments were carried out at equal multiplicity of infection in activated T cells and monocyte-derived dendritic cells (MO-DC) in parallel. Results Dual infection/competition experiments in activated T cells clearly indicated that CRF02_AG isolates had a significant replication advantage over the subtype A and subtype G viruses. The higher fitness of CRF02_AG was evident for isolates from patients with CD4+ T cell counts >200 cells/μL (non-AIDS) or CD4+ T cell counts Conclusion We observed a higher ex vivo replicative fitness of CRF02_AG isolates compared to subtype A and G viruses from the same geographic region and showed that this was independent of the co-receptor tropism and irrespective of high or low CD4+ T cell count. This advantage in replicative fitness may contribute to the dominant spread of CRF02_AG over A and G subtypes in West and West Central Africa.

To study recombination and the natural polymorphism in pol of HIV-1 strains in the Democratic Republic of Congo (DRC) we sequenced the protease and RT genes for 70 HIV-1 strains previously characterized in the env V3-V5 region from a sentinel surveillance study in 2002. For 41 of the 70 (58.6%) strains, the same subtype/ CRF designations were observed in pol and env. Twenty-three (32.9%) of 70 pol sequences were complex recombinants involving two to five subtypes as well as fragments that could not be classified into any of the known subtypes. All subtypes were involved in recombination events. Unclassified (U) and env subtype H strains were very likely to be recombinant strains. Overall, many minor mutations were identified in the protease sequences. Although at the time of our study ARV use was not yet widespread in DRC, three strains were identified with one major mutation associated with drug resistance: L90M and M46L in protease and K103N in RT.

Phylogenetic analysis on partial env sequences of HIV-1-positive samples obtained from sentinel population groups in HIV serosurveillance studies in 1997 and 2002 in the Democratic Republic of Congo (DRC) revealed a high genetic diversity in the number of cocirculating subtypes/CRFs as well as intrasubtype diversity. A cluster of three strains that formed a distinct and well-separated group within the subtype A radiation was observed. To determine a more precise structure of these viruses, the full-length genomes were sequenced. Phylogenetic tree and bootscan analysis showed that all three newly characterized viruses formed a new nonrecombinant lineage that was more closely related to subtype A. The SUDI (Subtype Distance) program showed that the distances of the newly derived HIV-1 sequences to subtype A references fell in the range of distances previously characterized for subsubtypes. According to current nomenclature rules, we have thus designated this new lineage as subsubtype A4. Analysis of viral sequences from other African countries suggests that the A4 strains seem restricted to DRC where they already circulated at the onset of the HIV/AIDS epidemic.

To document the actual genetic diversity of HIV-1 strains in the Republic of Congo, 114 HIV-1 positives persons were sampled in 2003 and 2004 after their informed consent. They were attending the teaching hospital, the reference health center in Makelekele, Brazzaville and the regional hospital centers in Pointe-Noire, Gamboma and Ouesso. A total of 104 samples were genetically characterized by direct sequencing of the p24 gag region and 80 were also subtyped in the V3-V5 env region. The genetic subtype distribution of the Congolese strains showed the predominance of subtype A (36.5% and 32.5% in gag and env, respectively) and G (30.8% and 21.25%), whereas subtype D strains represented 12.5% and 15%. Subtypes C, F, H, J, K and the CRFs-01, -02, -05 -06, and also the recently characterized CRF18 were seen at lower rates. Finally, 4.8% (gag) and 6.25% (env) of the strains could not be classified. Moreover, a high intra-subtype diversity was observed in our study. Among 70 strains which have been characterized in the two genomic regions, 14 (20%) appeared to be unique recombinants. These data show a high genetic variability in the Republic of Congo, where all the subtypes have been documented together with certain subsubtypes and several CRFs. (c) 2006 B.V. All rights reserved.

Based on partial env and pol (protease and RT) subtyping, we recently documented that the majority (>80%) of the HIV-1 strains that circulate in Cote d'Ivoire were CRF02_AG and about 11% were recombinants or could not be clearly assigned to a known subtype or CRF. In order to determine in more detail the precise structure of these viruses we sequenced the full-length genomes for six such strains. Bootscan and phylogenetic tree analysis showed that four strains were complex and unique CRF02_AG/CRF09_cpx recombinants, one was a CRF02_AG/CRF06_cpx recombinant, and one was a pure CRF09_cpx. Reanalysis of the remaining recombinants asserted the predominance of CRF09_cpx within intersubtype recombinants and circulation of CRF09_cpx in Cote d'Ivoire. More detailed analysis of the CRF09_cpx strains revealed also that part of the pol gene belonged to subtype K. This is the first time that such recombinants are described.

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.

No data on HIV-1 genetic subtypes in Burkina Faso have yet been reported. Among 70 samples obtained in 2001, the predominant env subtype was CRF06-cpx, followed by CRF02-AG, subtypes A and G. Nineteen of the 70 samples from 2001 had discordant subtype or circulation recombinant form (CRF) designations between env and gag. In contrast to surrounding countries, CRF06 is predominant in Burkina Faso. This virus was probably introduced early in the HIV epidemic because of its high representation in strains tested in 1996.

The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in the env V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations between env and gag . Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups : F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propse to divide subtype F into two subclades, with F1 gathering the known subtype strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains. (Résumé d'auteur)

In this paper, we studied the variability of HIV-1 subtype F strains in Africa. For 11 viruses, mainly of Central African origin, different parts of the genome were genetically characterized. For all strains the V3–V5 region of the envelope gene was sequenced, and for 7 strains, the entire envelope gene was studied. For 10 strains, the p24 region of the gag gene was also sequenced. For each region studied, three subgroups in the F subtype were identified, F1, F2, and F3. These three subgroups were supported by high bootstrap values and the intra- and inter-subgroup F distances were comparable to those obtained for the known subtypes A, B, C, D, E, G, and H. In subgroup F1, some African strains clustered with previously described strains from Brazil and Romania, suggesting an African origin of the HIV-1 epidemic in these countries. A more detailed analysis of the gag and the envelope sequences allowed the identification of four recombinant viruses. Our data show a high diversity among subtype F strains, suggesting the presence of new subtypes in the regions studied. If biological differences exist among subtypes, it is important that these subtypes be well defined. The data from our study show that there is a need to clearly identify the different subgroups within the F subtype.

High simian immunodeficiency virus (SIV) seroprevalence rates have been reported in the different African green monkey (AGM) subspecies. Genetic diversity of these viruses far exceeds the diversity observed in the other lentivirus-infected human and nonhuman primates and is thought to reflect ancient introduction of SIV in the AGM population. We investigate here genetic diversity of SIVagm in wild-living AGM populations from the same geographical locale (i.e., sympatric population) in Senegal. For 11 new strains, we PCR amplified and sequenced two regions of the genome spanning the first tat exon and part of the transmembrane glycoprotein. Phylogenetic analysis of these sequences shows that viruses found in sympatric populations cluster into distinct lineages, with at least two distinct genotypes in each troop. These data strongly suggest an ancient introduction of these divergent viruses in the AGM population.