Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa

Abstract

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.

FIG. 1

Distribution of HIV-1 env genetic subtypes in different geographic locales from the DRC.

FIG. 2

Phylogenetic tree based on 441 unambiguously aligned nucleotides from the env V3-V5 region of the 197 new HIV-1 isolates, from Kinshasa (a) and from Bwamanda and Mbuyi-Mayi (b), and reference strains representing the different genetic subtypes: A-KE.Q2317, A-SE.SOSE7253, A-92UG037, CRF02-AG-IBNG, CRF02-AG-DJ263, CRF02-AG-DJ264, B-RF, B-WEAU160, B-JRFL, B-HBX2, C-ETH2220, C-92BR025, C-IN.21068, C-BW.96BW0502, D-NDK, D-ZR.84ZR085, D-94UG114, D-ELI, CRF01-AE-90CR402, CRF01-AE-93TH253, CRF01-AE-CM240, F1-93BR020, F1-BZ163, F1-BZ126, F1-BE.VI850, F1-FI.FIN6393, F2-95CMMP255, F2-95CMMP257, G-BE.DRCBL, G-HH8793, G-SE6165, H-90CF056, H-BE.VI991, H-BE.VI997, J-SE.SE91733, J-SE.SE92809, K-96CMMP535, and K-97ZREQTB11. The analysis was performed as described in Materials and Methods. Reference strains are in grey, and strains from the DRC are indicated in black. Non-rec, nonrecombinant.

FIG. 3

Intra- and intersubtype genetic distances in the env V3-V5 region of HIV-1 isolates from the DRC. Range of lowest to highest distance within a subtype is in parentheses. A/E, CRF01-AE.

FIG. 4

Phylogenetic tree based on 621 unambiguously aligned nucleotides from the gag p24 region of the 62 new HIV-1 isolates and reference strains representing different genetic subtypes. The analysis was performed as described in Materials and Methods, and the same reference strains as in Fig. 2 were used. Reference strains are in grey, and strains from DRC are indicated in black.

FIG. 5

(a) Unrooted phylogenetic tree based on 2,278 unambiguously aligned nucleotides from the entire gp160 gene from 13 new HIV-1 isolates and reference strains representing different genetic subtypes. Analysis was performed as described in Materials and Methods; the reference strains used in the phylogenetic tree analysis were A-KE.Q2317, A-SE.SOSE7253, A-92UG037, CRF02-AG-IBNG, CRF02-AG-DJ263, CRF02-AG-DJ264, B-WEAU160, B-JRFL, B-HBX2, C-ETH2220, C-IN.21068, C-BW.96BW0502, D-NDK, D-ZR.84ZR085, D-94UG114, CRF01-AE-90CR402, CRF01-AE-93TH253, CRF01-AE-CM240, F1-93BR020, F1-BE.VI850, F1-FI.FIN6393, F2-95CMMP255, F2-95CMMP257, G-BE.DRCBL, G-HH8793, G-SE6165, H-90CF056, H-BE.VI991, H-BE.VI997, J-SE.SE91733, J-SE.SE92809, K-96CMMP535, and K-97ZREQTB11. (b) Putative recombination breakpoints within the gp160 gene were localized based on DIVERT and bootscan analysis (see Materials and Methods) using the reference strains listed above. The results were confirmed by phylogenetic tree analysis with 1,000 bootstrap replicates. Subtypes were designated when the bootstrap values were above 800, divergent subtypes were designated when bootstrap values were between 650 and 800, and fragments were identified as unclassified when they did not cluster at all with any of the known subtypes.