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Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors.

Pessôa R, Loureiro P, Esther Lopes M, Carneiro-Proietti AB, Sabino EC, Busch MP, Sanabani SS - PLoS ONE (2016)

Bottom Line: To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped.Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Department of Pathology, LIM 03, Hospital das Clínicas (HC), School of Medicine, University of São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).

Materials and methods: A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.

Results: Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1).

Conclusion: Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.

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Related in: MedlinePlus

Schematic representation of the NFLGs and breakpoint profiles of the recombinant sequences from proviral DNA are generated by a deep sequencing approach identified in this study.Sequences were mapped relative to the HXB2 numbering system. HIV subtypes are color codes and indicated at the bottom. CRF70_BF1 variants are boxed with red rectangle squares, CRF71_BF1 with blue rectangle squares, and CRF72_BF1 with orange rectangle squares.
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pone.0152499.g003: Schematic representation of the NFLGs and breakpoint profiles of the recombinant sequences from proviral DNA are generated by a deep sequencing approach identified in this study.Sequences were mapped relative to the HXB2 numbering system. HIV subtypes are color codes and indicated at the bottom. CRF70_BF1 variants are boxed with red rectangle squares, CRF71_BF1 with blue rectangle squares, and CRF72_BF1 with orange rectangle squares.

Mentions: Among the 59 NFLG isolates with strong evidence of recombination, 51 (86.4%) were unique mosaic isolates consisting of subtype B and F1 (n = 32), CRF70_BF1 (n = 4), CRF71_BF1 (n = 11), and CRF72_BF1 (n = 4). The remaining eight sequences belonged to four different HIV-1 recombinant subtypes and all contained fragments of subtype C in their genomes (Fig 3). The genetic heterogeneity is remarkable in RJ where at least four different patterns of recombination were observed. As shown in Fig 3, two distinct BF1 recombinant profiles, with four samples in profile I and nine in profile II were detected in samples from PE. These sequences were registered with the Los Alamos National Database as CRF70_BF1 (red rectangle box) and CRF71_BF1 (blue rectangle box). Detailed information of these sequences has recently been published by our group [26]. One BF1 sequence from SP (10BR_SP026) displayed a recombinant structure identical to the newly described CRF71_BF1. Recombination analyses of sequences from MG show four sequences with identical recombinant profile essentially consisted of subclades F1 and subtype B as parental sequences (green rectangle box, Fig 3) and appeared different from all previously documented CRFs in Brazil and South America. These sequences have recently been identified as CRF72_BF1 [26]. According to our estimate, the new CRF72_BF1 accounts for 9.7% (4/41) of the HIV-1 circulating strains among blood donors in MG and is responsible for more than 44% (4/9) of infections caused by all recombinant variants circulating in that region. To further identify more HIV-1 sequences with a similar recombinant structure, all NFLGs URFs and CRFs of BF1, BC, CF1, and BCF1 sequences were retrieved from Los Alamos database and analyzed together with our unique recombinant NFLG sequences. The results revealed no evidence of a relationship with any previously reported variants and thus these sequences are representing novel HIV-1 unique recombinants.


Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors.

Pessôa R, Loureiro P, Esther Lopes M, Carneiro-Proietti AB, Sabino EC, Busch MP, Sanabani SS - PLoS ONE (2016)

Schematic representation of the NFLGs and breakpoint profiles of the recombinant sequences from proviral DNA are generated by a deep sequencing approach identified in this study.Sequences were mapped relative to the HXB2 numbering system. HIV subtypes are color codes and indicated at the bottom. CRF70_BF1 variants are boxed with red rectangle squares, CRF71_BF1 with blue rectangle squares, and CRF72_BF1 with orange rectangle squares.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816342&req=5

pone.0152499.g003: Schematic representation of the NFLGs and breakpoint profiles of the recombinant sequences from proviral DNA are generated by a deep sequencing approach identified in this study.Sequences were mapped relative to the HXB2 numbering system. HIV subtypes are color codes and indicated at the bottom. CRF70_BF1 variants are boxed with red rectangle squares, CRF71_BF1 with blue rectangle squares, and CRF72_BF1 with orange rectangle squares.
Mentions: Among the 59 NFLG isolates with strong evidence of recombination, 51 (86.4%) were unique mosaic isolates consisting of subtype B and F1 (n = 32), CRF70_BF1 (n = 4), CRF71_BF1 (n = 11), and CRF72_BF1 (n = 4). The remaining eight sequences belonged to four different HIV-1 recombinant subtypes and all contained fragments of subtype C in their genomes (Fig 3). The genetic heterogeneity is remarkable in RJ where at least four different patterns of recombination were observed. As shown in Fig 3, two distinct BF1 recombinant profiles, with four samples in profile I and nine in profile II were detected in samples from PE. These sequences were registered with the Los Alamos National Database as CRF70_BF1 (red rectangle box) and CRF71_BF1 (blue rectangle box). Detailed information of these sequences has recently been published by our group [26]. One BF1 sequence from SP (10BR_SP026) displayed a recombinant structure identical to the newly described CRF71_BF1. Recombination analyses of sequences from MG show four sequences with identical recombinant profile essentially consisted of subclades F1 and subtype B as parental sequences (green rectangle box, Fig 3) and appeared different from all previously documented CRFs in Brazil and South America. These sequences have recently been identified as CRF72_BF1 [26]. According to our estimate, the new CRF72_BF1 accounts for 9.7% (4/41) of the HIV-1 circulating strains among blood donors in MG and is responsible for more than 44% (4/9) of infections caused by all recombinant variants circulating in that region. To further identify more HIV-1 sequences with a similar recombinant structure, all NFLGs URFs and CRFs of BF1, BC, CF1, and BCF1 sequences were retrieved from Los Alamos database and analyzed together with our unique recombinant NFLG sequences. The results revealed no evidence of a relationship with any previously reported variants and thus these sequences are representing novel HIV-1 unique recombinants.

Bottom Line: To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped.Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Department of Pathology, LIM 03, Hospital das Clínicas (HC), School of Medicine, University of São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).

Materials and methods: A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.

Results: Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1).

Conclusion: Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.

Show MeSH
Related in: MedlinePlus