Sequencing and phylogenetic analysis of near full-length HIV-1 subtypes A, B, G and unique recombinant AC and AD viral strains identified in South Africa

Abstract

By the end of 2012, more than 6.1 million people were infected with HIV-1 in South Africa. Subtype C was responsible for the majority of these infections and more than 300 near full-length genomes (NFLGs) have been published. Currently very few non-subtype C isolates have been identified and characterized within the country, particularly full genome non-C isolates. Seven patients from the Tygerberg Virology (TV) cohort were previously identified as possible non-C subtypes and were selected for further analyses. RNA was isolated from five individuals (TV047, TV096, TV101, TV218, and TV546) and DNA from TV016 and TV1057. The NFLGs of these samples were amplified in overlapping fragments and sequenced. Online subtyping tools REGA version 3 and jpHMM were used to screen for subtypes and recombinants. Maximum likelihood (ML) phylogenetic analysis (phyML) was used to infer subtypes and SimPlot was used to confirm possible intersubtype recombinants. We identified three subtype B (TV016, TV047, and TV1057) isolates, one subtype A1 (TV096), one subtype G (TV546), one unique AD (TV101), and one unique AC (TV218) recombinant form. This is the first NFLG of subtype G that has been described in South Africa. The subtype B sequences described also increased the NFLG subtype B sequences in Africa from three to six. There is a need for more NFLG sequences, as partial HIV-1 sequences may underrepresent viral recombinant forms. It is also necessary to continue monitoring the evolution and spread of HIV-1 in South Africa, because understanding viral diversity may play an important role in HIV-1 prevention strategies.

FIG. 1.

Maximum likelihood (ML) tree inference of the near full-length genome (NFLG) concatenated sequence alignment. The ML tree inferred in phylogenetic analysis (phyML) contains the seven newly sequenced Tygerberg Virology (TV) isolates, HIV-1 reference strains, and 13 previously characterized non-subtype C strains from South Africa. The evolutionary distances were computed using the GTR model of nucleic acid substitution with an estimated Gamma shape parameter. The genetic distance is displaying in the scale bar at the bottom of the figure, while the major different clades of HIV-1 Group M have been highlighted. The sequence IDs of South African strains have been marked with the 7 newly genotyped isolates marked in red and the 13 previously genotyped isolates marked in blue. Bootstrap support values for the internal branches for each major clade are shown with an asterisk and indicate support higher than 90%. Color images available online at www.liebertpub.com/aid

FIG. 2.

Unique recombinant form of TV101. (A) Bootscan similarity plot constructed in SimPlot v. 3.5.1 shows the recombinant profile of this viral isolate. (B) Schematic diagram of A1 and D recombinant segments of TV101. ML trees of TV101 fragments with complimentary reference sequences were inferred in phyML with a total of 1,000 bootstrap replicates. The evolutionary distances were computed using the GTR model of nucleic acid substitution with an estimated Gamma shape parameter. Each roman numeral in the similarity plot at the top corresponds to a phylogeny in the bottom part of the schematic. TV101 is marked in red in each of the seven different phylogenies, while bootstrap support values for each of the clades in which the isolate clusters are also shown in red. Color images available online at www.liebertpub.com/aid

FIG. 3.

Unique recombinant form of TV218. (A) Bootscan similarity plot constructed in SimPlot v. 3.5.1 shows the recombinant profile of this viral isolate. (B) Schematic diagram of A1 and C recombinant segments of TV218. Radiation maximum trees of TV218 fragments with complimentary reference sequences were implemented in phyML with a total of 1,000 bootstrap replicates. The evolutionary distances were computed using the GTR model of nucleic acid substitution with an estimated Gamma shape parameter. Each roman numeral in the similarity plot in the top corresponds to a phylogeny in the bottom part of the schematic. TV218 is marked in red in each of the four different phylogenies, while bootstrap support values for each of the clades in which the isolate clusters are also shown in red. Color images available online at www.liebertpub.com/aid

FIG. 4.

jpHMM analysis of South African AC recombinants. (A) Indication of the recombinant breakpoints based on HXB2 numbering. The uncolored regions denote missing information due to the input fragment sequence and the gray regions denote missing information due to uninformative subtype models. (B) Posterior probabilities of the subtypes at each sequence position (original sequence positions) calculated by jpHMM. Color images available online at www.liebertpub.com/aid