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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.

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.

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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 propose to divide subtype F into two subclades, with F1 gathering the known subtype F 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.

IntroductionAntiretroviral treatment (ART) has been scaled up over the last decade but compared to adults, children living with HIV are less likely to receive ART. Moreover, children and adolescents are more vulnerable than adults to virological failure (VF) and emergence of drug resistance. In this study we determined virological outcome in perinatally HIV-1-infected children and adolescents receiving ART in Togo.MethodsHIV viral load (VL) testing was consecutively proposed to all children and adolescents who were on ART for at least 12 months when attending HIV healthcare services for their routine follow-up visit (June to September 2014). Plasma HIV-1 VL was measured using the m2000 RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA). Genotypic drug resistance was done for all samples with VL>1000 copies/ml.Results and discussionAmong 283 perinatally HIV-1-infected children and adolescents included, 167 (59%) were adolescents and 116 (41%) were children. The median duration on ART was 48 months (interquartile range: 28 to 68 months). For 228 (80.6%), the current ART combination consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) (zidovudine and lamivudine) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine or efavirenz). Only 28 (9.9%) were on a protease inhibitor (PI)-based regimen. VL was below the detection limit (i.e. 40 copies/ml) for 102 (36%), between 40 and 1000 copies/ml for 35 (12.4%) and above 1000 copies/ml for 146 (51.6%). Genotypic drug-resistance testing was successful for 125/146 (85.6%); 110/125 (88.0%) were resistant to both NRTIs and NNRTIs, 1/125 (0.8%) to NRTIs only, 4/125 (3.2%) to NNRTIs only and three harboured viruses resistant to reverse transcriptase and PIs. Overall, 86% (108/125) of children and adolescents experiencing VF and successfully genotyped, corresponding thus to at least 38% of the study population, had either no effective ART or had only a single effective drug in their current ART regimen.ConclusionsOur study provided important information on virological outcome on lifelong ART in perinatally HIV-1-infected children and adolescents who were still on ART and continued to attend antiretroviral (ARV) clinics for follow-up visits. Actual conditions for scaling up and monitoring lifelong ART in children in resource-limited countries can have dramatic long-term outcomes and illustrate that paediatric ART receives inadequate attention.

Members of HIV-1 group M are responsible for the vast majority of AIDS cases worldwide and have been classified on the basis of their phylogenetic relationships into nine roughly equidistant clades, termed subtypes. Although there are no known phenotypic correlates for these genotypes, the disproportionate spread of certain of these lineages has been taken to indicate that subtype-specific biological differences may exist. The subtype nomenclature thus remains an important molecular epidemiological tool with which to track the course of the group M pandemic. In this study, we have characterized HIV-1 strains described previously as unusual subtype A variants on the basis of partial sequence analysis. Six such strains from Cyprus (CY), South Korea (KR), and the Democratic Republic of Congo (CD) were PCR amplified from infected cell culture or patient PBMC DNA, cloned, and sequences in their entirety (94CY017, 97KR004, 97CDKTB48, and 97CDKP58) or as half genomes (97CDKS10 and 97CDKFE4). Distance and phylogenetic analyses showed that four of these viruses (94CY017, 97CDKTB48, 97CDKFE4, and 97CDKS10) were closely related to each other, but quite divergent from all other HIV-1 strains, except for subtype A viruses, which represented their closest relatives. In phylogenetic trees from gag, pol, env, and nef regions, the four newly characterized HIV-1 strains formed a distinct sister clade to subtype A, which was as closely related to subtype A as subsubtypes F1 and F2 are to each other. According to current nomenclature rules, this defines a subsubtype, which we have tentatively termed A2. The two other viruses, 97KR004 and 97CDKP58, as well as a full-length HIV-1 sequence from the sequence database (ZAM184), were found to represent complex A2/D, A2/G, and A2/C recombinants, respectively. These results indicate that HIV-1 subtype A is composed of two subsubtypes (A1 and A2), both of which appear to have a widespread geographic distribution. The A2 viruses described here represent the first reference reagents for this new group M lineage.

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