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Type-1 HIV (HIV-1) group M (HIV-1M) genetic diversity is highest in the Congo Basin where the epidemic ignited a century ago. HIV-1M has diversified into multiple subtypes, sub-subtypes, and circulating and unique recombinant forms (CRFs/URFs). An unanswered question is why some rare subtypes never reached epidemic levels despite their age. Several studies identified the role of HIV-1M accessory genes nef and vpu in virus adaptation to human hosts and subsequent spread. Other reports also pointed out the pivotal role of gag in transmissibility, virulence, and replication capacity. In this study we characterized the HIV-1 gag gene of 148 samples collected in different localities of the Democratic Republic of the Congo (DRC) between 1997 and 2013. We used nested polymerase chain reaction (PCR) to amplify the whole gag gene. PCR products were sequenced either by Sanger method or by next generation sequencing on Illumina MiSeq or iSeq100 platforms. Generated sequences were used for subsequent analyses using different bioinformatic tools. Phylogenetic analysis of the generated sequences revealed a high genetic diversity with up to 22 different subtypes, sub-subtypes, CRFs. Up to 15% (22/148) URFs were identified, in addition to rare subtypes such as H, J, and K. At least two amino acid motifs present in the gag gene have been shown to modulate HIV-1 replication, budding, and fitness: the P(T/S)AP and the LYPXnL motifs. Structural analysis revealed the presence of P(T/S)AP in all the 148 sequences with the majority (136/148) bearing the PTAP. Three samples presented a duplication of this motif. The LYPXnL motif was identified in 38 of 148 sequences. There was no clear link between the frequency of these motifs and HIV-1M subtypes. In summary, we confirmed a high genetic diversity of HIV-1M in the DRC. We observed the presence of amino acid motifs important for viral replication and budding even in some rare HIV-1 subtypes. Their impact on viral fitness needs be further evaluated by in vitro studies.

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.

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.

Abstract In this study, we characterized four HIV-1 strains from Cameroon, Gabon, and the Democratic Republic of Congo (DRC), collected during independent serosurveys, and previously found to cluster in the pol gene with HIV-1 MAL and HIV-1 NOGIL3, two complex recombinant viruses reported in the early HIV epidemic, and with the recombinant strain 04FR.AUK recently described in France. The four newly sequenced viruses shared the same structure as 04FR.AUK, involving alternating fragments of subtype A, K, and unclassified (U) fragments, representing a new CRF called CRF45_AKU. Some of the unclassified fragments were related to unclassified regions described in either CRF04 or CRF09 strains. Careful reanalysis of HIV-1 MAL and HIV-1 NOGIL3 demonstrated that these strains were related exclusively to CRF45_AKU and either two subtype D fragments for HIV-1 MAL or one subtype H segment for HIV-1 NOGIL3. Following extensive blast searches, related gag, pol, and env sequences were observed in Central and West Africa (Senegal, Mali), as well as in Europe (France, Spain, Italy, Cyprus), Argentina, and China.

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.

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.

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.

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.

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 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.

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 Côte 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 Côte 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.

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.

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.

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.

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.