Molecular cloning and phylogenetic analysis of human immunodeficiency virus type 1 subtype C: a set of 23 full-length clones from Botswana

Abstract

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.

FIG. 1

Phylogenetic relationship of the newly characterized full-length clones from Botswana (boxed in black) to other representative full-length HIV-1 sequences of subtypes A, B, C, D, F, and H and recombinant subtypes AE, AG, and BF. Full-length subtype C sequences from India were also included in the analysis. A neighbor-joining tree was constructed on the basis of the hidden Markov model nucleotide alignment of full-length HIV-1 genomes. Subtype O ANT70 sequence was used as an outgroup. Values along the branches indicate the bootstrap values that support branching (out of a 1,000 resampling).

FIG. 2

Variability plots comparing subtype B and C (Botswana) sequences across the entire HIV-1 genome. The variability distribution was estimated as an entropy function of the nucleotide variation in the SWAN program based on the hidden Markov model alignment of the complete HIV-1 genome. The subtype B sequences used in the analysis were DH123, 89.6, RF, WEAU, OYI, HXB2, JRFL, and YU2. n, number of sliding window sites across the HIV-1 genome with gap stripping.

FIG. 3

Intrasample diversity among subtype B (open bars) and newly derived subtype C (solid bars) clones from Botswana. Pairwise distances were computed by the DNADIST program from the PHYLIP package based on hidden Markov model alignment of the complete genome or individual genes or subgenomic regions (B) retrieved from the same alignment. Three sets of subtype B sequences were included: JRFL and FRCSF, YU2 and YU10, and ACH320A and ACH320B. Seven sets of subtype C Botswana clones were included: BW01, BW04, BW05, BW11, BW15, BW16, and BW17. The order of bars for each gene or region corresponds to the order in which the sets are mentioned. Dashed ovals depict low- and high-diversity groups.