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

Sanabani SS, Pessôa R, Soares de Oliveira AC, Martinez VP, Giret MT, de Menezes Succi RC, Carvalho K, Tomiyama CS, Nixon DF, Sabino EC, Kallas EG - PLoS ONE (2013)

Bottom Line: Of the 51 samples studied, the NFLGs and partial fragments of HIV-1 from 42 PBMCs and 25 plasma were successfully subtyped.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 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.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Research Laboratory (LIM 03), School of Medicine, University of São Paulo, São Paulo, Brazil. sabyem_63@yahoo.com

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.

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Related in: MedlinePlus

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.
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pone-0062552-g002: 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.

Mentions: Based on phylogenetic analysis, the NFLGs and partial proviral nucleotide sequences (n = 42) of the clinical HIV-1 isolates indicated that 22 (52.4%) patients were infected with HIV-1 subtype B, 16 (38.1%) were infected with a mosaic consisting of subtype BF1 and 4 (9.5%) were infected with sub-subtype F1 (Table 2). Of the total 25 plasma samples for which viral subtype was determined, 17 (68%) were classified as subtype B, 3 (12%) were sub-subtype F1, and 5 (20%) were BF1 recombinant viruses (Table 2). All chimeric viruses were unique according to their recombination profile, i.e., not assigned to any subtype or CRF (Figure 1). The relationships of the viral sequences from patients’ PBMCs to the sequences obtained from the corresponding RNA virus within the same regions were examined for each patient to assess the viral diversity in both compartments. The results revealed that all but one patient, 010BR_IMT_020, had plasma RNA and proviral DNA variation only ranging between 0–2.7% (Figure 2). These relations were further confirmed by phylogenetic analysis, which showed close branching as demonstrated in Figure 3. These findings may indicate that the primary infected PBMCs of these patients were likely the source of plasma circulating viral sequences however; more sophisticated genetic tests able to detect viral population structure are needed to confirm this conclusion. The observed differences in the percent nucleotide variations between proviruses and plasma free viruses in this group may reflect evolution that occurs during the initial phase of acute infection, before the therapeutic control of HIV-1 replication is established. Surprisingly, the intra-individual plasma and proviral sequence variation for patient 010BR_IMT_020 in the overlapped regions depicted in Figure 4 were 9.8% and 6.5%, respectively, indicating that the plasma viruses were derived from a population significantly distinct from those of the cellular sources in this 13 years old asymptomatic patient. This result is consistent with dual distinct variants of the same subtype being involved in establishing infection. Dual infection with subclade F1 and BF1 recombinant was observed in patient 010BR_IMT_041 plasma sample (Figure 5). This patient was a nine year old child who diagnosed in February 2005 and until the sampling period had been asymptomatic. The patient had been receiving ART since September 2005. This observation of dual infection occurred accidentally during assembling of the generated data, in which some sequences failed to assemble to other overlapping stretches of fragment B1. As a result, we sought to compare this stretch to HIV sequences available from public databases. Upon analysis with the basic local alignment search tool (BLAST) available from GenBank, the stretch (010BR_IMT_041_PL- REC; 548 bp) from plasma revealed high percentages of nucleotide sequence identity to the BF1 isolate 99JY-TRA0133 (Genbank accession: JN235964), whereas the other larger fragment (010BR_IMT_041_pl; 5720 bp) revealed high homology to subclade F1 isolate 02BR082 (Genbank accession: FJ771006) at the nucleotide levels. To ensure that the generation of the two consensus sequences from patient 010BR_IMT_041 plasma sample was not the result of sample contamination, repeat sequence analysis using the purified B1 amplicon was performed and revealed identical findings. These results possibly indicate that some internal sequencing primers of fragment B1 preferentially annealed to the BF1 string during sequencing reaction. Regions that were the same F1 subclade in the two pols were then compared to determine whether the 010BR_IMT_041_PL viruses were the actual parents of the recombinant fragment or if an infection in this patient was acquired with two genetically distinct viruses (Figure 5B). While both partial pol genes were sub-subtype F1 fragments, these were from different subclade F1 isolates, since the sequences from the two plasma demonstrated high nucleotide divergence (up to 6.8%). Moreover, as shown in Figure 5B, both F1 non-recombinant sequences recovered from plasma and PBMC clustered separately (aLRT 100%) and the branch lengths separating them from the F1 fragment involved in the recombination event were typical for other sequences of unrelated F1 variants. The analysis was then extended to include isolates with non-overlapping fragments, namely 010BR_IMT_013 and 010BR_IMT_027, to determine whether the PBMC viruses were truly parental strains to those recovered from the plasma. For this purpose, the phylogenetic clustering profile of the non-overlapped fragments from both compartments were compared to a number of additional Brazilin subtype B and other HIV-1 reference sequences to increase our confidence in the analyses and provide a broader perspective. These results revealed the magnitude of aLRT value supporting the identical clustering of the plasma isolate 010BR_IMT_013_pl (Figure 6A) and the proviral 010BR_IMT_013_pr strains (Figure 6B) with the Brazilian subtype B BREPM 1040 and 05BR 1092 subtype B sequences (branch marked with green color). Based on these results, it is possible to assume that the primary infected PBMCs in this patient were likely the source of the plasma circulating viral sequences. However, this interpretation does not hold true when the analysis was applied to the plasma and proviral non-overlapping fragments of patient 010BR_IMT_027. The clustering profile to subtype B references and genetic distances as shown in Figure S1 were significantly different between both fragments in this patient indicating dual infection with distinct subtype B variants.


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

Sanabani SS, Pessôa R, Soares de Oliveira AC, Martinez VP, Giret MT, de Menezes Succi RC, Carvalho K, Tomiyama CS, Nixon DF, Sabino EC, Kallas EG - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646872&req=5

pone-0062552-g002: 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.
Mentions: Based on phylogenetic analysis, the NFLGs and partial proviral nucleotide sequences (n = 42) of the clinical HIV-1 isolates indicated that 22 (52.4%) patients were infected with HIV-1 subtype B, 16 (38.1%) were infected with a mosaic consisting of subtype BF1 and 4 (9.5%) were infected with sub-subtype F1 (Table 2). Of the total 25 plasma samples for which viral subtype was determined, 17 (68%) were classified as subtype B, 3 (12%) were sub-subtype F1, and 5 (20%) were BF1 recombinant viruses (Table 2). All chimeric viruses were unique according to their recombination profile, i.e., not assigned to any subtype or CRF (Figure 1). The relationships of the viral sequences from patients’ PBMCs to the sequences obtained from the corresponding RNA virus within the same regions were examined for each patient to assess the viral diversity in both compartments. The results revealed that all but one patient, 010BR_IMT_020, had plasma RNA and proviral DNA variation only ranging between 0–2.7% (Figure 2). These relations were further confirmed by phylogenetic analysis, which showed close branching as demonstrated in Figure 3. These findings may indicate that the primary infected PBMCs of these patients were likely the source of plasma circulating viral sequences however; more sophisticated genetic tests able to detect viral population structure are needed to confirm this conclusion. The observed differences in the percent nucleotide variations between proviruses and plasma free viruses in this group may reflect evolution that occurs during the initial phase of acute infection, before the therapeutic control of HIV-1 replication is established. Surprisingly, the intra-individual plasma and proviral sequence variation for patient 010BR_IMT_020 in the overlapped regions depicted in Figure 4 were 9.8% and 6.5%, respectively, indicating that the plasma viruses were derived from a population significantly distinct from those of the cellular sources in this 13 years old asymptomatic patient. This result is consistent with dual distinct variants of the same subtype being involved in establishing infection. Dual infection with subclade F1 and BF1 recombinant was observed in patient 010BR_IMT_041 plasma sample (Figure 5). This patient was a nine year old child who diagnosed in February 2005 and until the sampling period had been asymptomatic. The patient had been receiving ART since September 2005. This observation of dual infection occurred accidentally during assembling of the generated data, in which some sequences failed to assemble to other overlapping stretches of fragment B1. As a result, we sought to compare this stretch to HIV sequences available from public databases. Upon analysis with the basic local alignment search tool (BLAST) available from GenBank, the stretch (010BR_IMT_041_PL- REC; 548 bp) from plasma revealed high percentages of nucleotide sequence identity to the BF1 isolate 99JY-TRA0133 (Genbank accession: JN235964), whereas the other larger fragment (010BR_IMT_041_pl; 5720 bp) revealed high homology to subclade F1 isolate 02BR082 (Genbank accession: FJ771006) at the nucleotide levels. To ensure that the generation of the two consensus sequences from patient 010BR_IMT_041 plasma sample was not the result of sample contamination, repeat sequence analysis using the purified B1 amplicon was performed and revealed identical findings. These results possibly indicate that some internal sequencing primers of fragment B1 preferentially annealed to the BF1 string during sequencing reaction. Regions that were the same F1 subclade in the two pols were then compared to determine whether the 010BR_IMT_041_PL viruses were the actual parents of the recombinant fragment or if an infection in this patient was acquired with two genetically distinct viruses (Figure 5B). While both partial pol genes were sub-subtype F1 fragments, these were from different subclade F1 isolates, since the sequences from the two plasma demonstrated high nucleotide divergence (up to 6.8%). Moreover, as shown in Figure 5B, both F1 non-recombinant sequences recovered from plasma and PBMC clustered separately (aLRT 100%) and the branch lengths separating them from the F1 fragment involved in the recombination event were typical for other sequences of unrelated F1 variants. The analysis was then extended to include isolates with non-overlapping fragments, namely 010BR_IMT_013 and 010BR_IMT_027, to determine whether the PBMC viruses were truly parental strains to those recovered from the plasma. For this purpose, the phylogenetic clustering profile of the non-overlapped fragments from both compartments were compared to a number of additional Brazilin subtype B and other HIV-1 reference sequences to increase our confidence in the analyses and provide a broader perspective. These results revealed the magnitude of aLRT value supporting the identical clustering of the plasma isolate 010BR_IMT_013_pl (Figure 6A) and the proviral 010BR_IMT_013_pr strains (Figure 6B) with the Brazilian subtype B BREPM 1040 and 05BR 1092 subtype B sequences (branch marked with green color). Based on these results, it is possible to assume that the primary infected PBMCs in this patient were likely the source of the plasma circulating viral sequences. However, this interpretation does not hold true when the analysis was applied to the plasma and proviral non-overlapping fragments of patient 010BR_IMT_027. The clustering profile to subtype B references and genetic distances as shown in Figure S1 were significantly different between both fragments in this patient indicating dual infection with distinct subtype B variants.

Bottom Line: Of the 51 samples studied, the NFLGs and partial fragments of HIV-1 from 42 PBMCs and 25 plasma were successfully subtyped.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 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.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Research Laboratory (LIM 03), School of Medicine, University of São Paulo, São Paulo, Brazil. sabyem_63@yahoo.com

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.

Show MeSH
Related in: MedlinePlus