Variability of HIV-1 genomes among children and adolescents from São Paulo, Brazil

Abstract

Background: Genetic variability is a major feature of the human immunodeficiency virus type 1 (HIV-1) and considered the key factor to frustrating efforts to halt the virus epidemic. In this study, we aimed to investigate the genetic variability of HIV-1 strains among children and adolescents born from 1992 to 2009 in the state of Sao Paulo, Brazil.

Methodology: Plasma and peripheral blood mononuclear cells (PBMC) were collected from 51 HIV-1-positive children and adolescents on ART followed between September 1992 and July 2009. After extraction, the genetic materials were used in a polymerase chain reaction (PCR) to amplify the viral near full length genomes (NFLGs) from 5 overlapped fragments. NFLGs and partial amplicons were directly sequenced and data were phylogenetically inferred.

Results: Of the 51 samples studied, the NFLGs and partial fragments of HIV-1 from 42 PBMCs and 25 plasma were successfully subtyped. Results based on proviral DNA revealed that 22 (52.4%) patients were infected with subtype B, 16 (38.1%) were infected with BF1 mosaic variants and 4 (9.5%) were infected with sub-subtype F1. All the BF1 recombinants were unique and distinct from any previously identified unique or circulating recombinant forms in South America. Evidence of dual infections was detected in 3 patients coinfected with the same or distinct HIV-1 subtypes. Ten of the 31 (32.2%) and 12 of the 21 (57.1%) subjects with recovered proviral and plasma, respectively, protease sequences were infected with major mutants resistant to protease inhibitors. The V3 sequences of 14 patients with available sequences from PBMC/or plasma were predicted to be R5-tropic virus except for two patients who harbored an X4 strain.

Conclusions: The high proportion of HIV-1 BF1 recombinant, coinfection rate and vertical transmission in Brazil merits urgent attention and effective measures to reduce the transmission of HIV among spouses and sex partners.

Figure 1. Schematic representation of the NFLG, partial structure and breakpoint profiles of the BF1 sequences identified in this study both from HIV RNA and proviral DNA.

Samples that were identified in this study to host distinct viruses are indicated with the star symbol. The region of subclade F1 and subtypes B are indicated at the bottom.

Figure 2. Genetic distances of overlapping regions between isolates recovered from patients with paired plasma and PBMC samples.

Concatenated sequences are indicated with the star symbol. The region of subclade F1 and subtypes B are indicated at the bottom.

Figure 3. Maximum-likelihood phylogenetic trees form each non-recombinant fragment were constructed using all available sequences from proviral DNA (indicated by black circles) and plasma isolate (indicated by an empty circles) along with HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

The numbering for the HIV-1 fragment A, B1, B2 and C sequences corresponds to the HXB2 reference sequence. For purposes of clarity, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥70% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.

Figure 4. Genetic distances and phylogenetic tree constructed using a maximum-likelihood method from concatenated regions of HIV RNA (indicated by black circles) and proviral DNA (indicated by black squares) marked with Arabic numbers 1 and 2 from patient 010BR_IMT_020 along with HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

For purposes of clarity, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥70% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.

Figure 5. Genetic distances and phylogenetic tree constructed using a maximum-likelihood method of HIV isolates recovered from patient 01BR_IMT_041.

A: Phylogenetic tree from concatenated regions assigned as subtype B from the BF1 recombinant isolate. B: Phylogenetic tree showing the clustering pattern of F1 sequences (marked by dotted box). F1 region from genuine F1 sequence recovered from plasma and PBMCs are marked by a black circle and square, respectively while the F1 region from the BF1 recombinant sequence is marked by an empty square. The approximate likelihood ratio test (aLRT) values of ≥70% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.

Figure 6. Phylogenetic clustering profile of the non-overlapped fragments assigned as subtype B from both plasma and provirus isolate 01BR_IMT_013 were compared to a number of additional Brazilian subtype B sequences and other HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

For purposes of clarity, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥70% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.