Identification of New HIV-1 Circulating Recombinant Forms CRF81_cpx and CRF99_BF1 in Central Western Brazil and of Unique BF1 Recombinant Forms

Abstract

Intersubtype recombinants classified as circulating recombinant forms (CRFs) or unique recombinant forms (URFs) have been shown to play an important role in the complex and dynamic Brazilian HIV/AIDS epidemic. Previous pol region studies (2003-2013) in 828 patients from six states from Central Western, Northern and Northeastern Brazil reported variable rates of BF1, F1CB, BC, and CF1 mosaics. In this study HIV-1 subtype diversity BF1, F1CB, BC, and CF1 recombinants in pol were analyzed. Full/near-full/partial genome sequences were generated from F1CB and BF1 recombinants. Genomic DNA extracted from whole blood was used in nested-PCR to amplify four overlapping fragments encompassing the full HIV-1 genome. Phylogenetic trees were generated using the neighbor-joining/NJ method (MEGA 6.0). The time of the most recent common ancestor (TMRCA) of F1CB and BF1 clades was estimated using a Bayesian Markov Chain Monte Carlo approach (BEAST v1.8; BEAGLE). Bootscanning was used for recombination analyses (Simplot v3.5.1); separate NJ phylogenetic analysis of fragments confirmed subtypes. The phylogenetic analyses of protease/reverse-transcriptase sequences in 828 patients revealed 76% subtype B (n = 629), 6.4% subtype C (n = 53), 4.2% subtype F1 (n = 35), 13.4% intersubtype recombinants: 10.5% BF1 (n = 87), 2.3% BC (n = 19), 0.4% F1CB (n = 3), and 0.2% CF1 (n = 2). Two full and one partial BF1C genomes allowed the characterization of the CRF81_cpx that has 9 breakpoints dividing the genome into 10 subregions. Basic Local Alignment Search Tool searches (Los Alamos HIV Sequence Database) identified six other sequences with the same recombination profile in pol, five from Brazil, and one from Italy. The estimated median TMRCA of CRF81_cpx was 1999 (1992-2003). CRF60_BC-like sequences, originally described in Italy, were also found. Two full and one near full-length BF1 genomes led to the characterization of the new CRF99_BF1 that has six recombination breakpoints dividing the genome into seven subregions. Two new URFs BF1, with six recombination breakpoints and seven subregions were also characterized. The description of the first Brazilian BF1C CRF81_cpx and of the new CRF99_BF1 corroborate the important role of CRFs in the HIV/AIDS epidemic throughout Brazil. Our data also highlight the value of HIV-1 full-genome sequence studies in order to fully reveal the complexity of the epidemic in a huge country as Brazil.

Keywords: Brazil; CRFs; Central Western; HIV-1; URFs; molecular epidemiology.

FIGURE 1

Map of Brazil highlighting our study area. The pie-charts depict the frequency of HIV-1 subtypes of 828 patients living in six states located in three geographic regions, Central Western: Goiás/GO, Mato Grosso/MT, Mato Grosso do Sul/MS; Northern: Tocantins/TO; and Northeastern: Maranhão/MA, Piauí/PI. The number of pol sequences analyzed is depicted below each pie chart.

FIGURE 2

Phylogenetic tree of partial HIV-1 pol sequences of F1CB, BC, and CF1 recombinants from Central Western, Northern, and Northeastern Brazil. The tree was constructed using MEGA software, 6.0 version under NJ method and Kimura two-parameter model (Bootstrap value over 80%). The mosaic patterns of recombinants: Cluster F1CB, Clusters BC (I and II), CRF31_BC-like, CRF60_BC-like, and Cluster CF1 are depicted. In the mosaic structure representations of F1CB, BC, and CF1 clusters, the breakpoint positions are indicated according to HXB2 genome position. In the mosaic structure, the green color stands for HIV-1 subtype F1, blue color stands for subtype B, and brown color stands for subtype C.

FIGURE 3

Phylogenetic tree including the three F1CB sequences identified here and the six pol sequences sharing over 95% similarity recovered from the BLAST search. The tree was constructed using MEGA software, 6.0 version under NJ method and Kimura two-parameter model (Bootstrap value over 80%).

FIGURE 4

Time-scaled Bayesian MCMC tree of nine pol sequences of F1CB HIV-1 sequences.

FIGURE 5

Mosaic structure of the CRF81_cpx composed of HIV-1 subtypes B, F1, and C. (A) Bootscanning analysis was conducted using a window size of 300 bp and a step size of 20 bp along with reference strains of representative subtypes B, F1, and C from HIV-1 group M. (B) Genomic structure of CRF81_cpx. Breakpoint positions according to HXB2 genome positions are indicated. The blue color stands for HIV-1 subtype B, green color stands for subtype F1, and brown color stands for subtype C. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

FIGURE 6

Phylogenetic analysis of five pol BF1 sequences with similar recombination profile. NJ method and Kimura two-parameter evolutionary model/1,000 replicate bootstrap values were applied. In the mosaic structure, the green color stands for subtype F1 and the blue color stands for subtype B.

FIGURE 7

Mosaic structure of the new CRF99_BF1. (A) Bootscanning analysis was conducted using a window size of 300 bp and a step size of 20 bp along with reference strains of B, C, and F1 representative HIV-1M subtypes. (B) Genomic structure of the CRF99_BF1. Breakpoint positions according to HXB2 genome numbering system are indicated. The blue color stands for subtype B, the green color stands for subtype F1, and the brown color for subtype C. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

FIGURE 8

Recombination breakpoint analyses of URF_BF1/BRGOMI744. (A) Bootscanning analysis was conducted using a window size of 300 bp and a step size of 20 bp along with reference strains of B, C, and F1 representative HIV-1 M subtypes. (B) Genomic structure of URF/BRGOMI744. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

FIGURE 9

Recombination breakpoint analyses of the URF_BF1 identified in the BRMT1319 strain. (A) Bootscanning analysis was conducted using a window size of 300 bp and a step size of 20 bp along with reference strains of B, C, and F1 representative HIV-1 M subtypes. (B) Genomic structure of the URF_BF1 identified in the BRMT1319 strain. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).