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

Pessôa, Rodrigo; Loureiro, Paula; Lopes, Maria Esther; Carneiro-Proietti, Anna Bárbara F.; Sabino, Éster Cerdeira; Busch, Michael P.; Sanabani, Sabri Saeed

doi:10.1371/journal.pone.0152499

Cited by 28 publications

(47 citation statements)

References 38 publications

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“…Among the 14 CRFs_BF1 described so far, eight originated in Brazil (CRF28_BF, CRF29_BF, CRF39_BF, CRF40_BF, CRF46_BF, CRF70_BF, CRF71_BF and CRF72_BF) [18–22]. The importance of BF1 recombinants in Brazil is further corroborated by the description of countless URFs in all country regions [23–26]. Previous studies from our research group in different study populations in Central West, North and Northeast Brazilian States showed variable prevalence of BF1 recombinants in the pol subgenomic fragment: (Goiás: 3.7–18.1%, Mato Grosso: 11.9%, Mato Grosso do Sul: 8.2–25.9%, Tocantins: 7.7%, Maranhão: 7.5%, Piauí: 4.5% [11,27–37].…”

Section: Introductionmentioning

confidence: 98%

Characterization of HIV-1 CRF90_BF1 and putative novel CRFs_BF1 in Central West, North and Northeast Brazilian regions

Reis¹,

Bello

Guimarães

et al. 2017

PLoS ONE

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The Brazilian AIDS epidemic has been characterized by an increasing rate of BF1 recombinants and so far eight circulating recombinant forms/CRFs_BF1 have been described countrywide. In this study, pol sequences (protease/PR, reverse transcriptase/RT) of 87 BF1 mosaic isolates identified among 828 patients living in six Brazilian States from three geographic regions (Central West, North, Northeast) were analyzed. Phylogenetic and bootscan analyses were performed to investigate the evolutionary relationship and mosaic structure of BF1 isolates. Those analyses showed that 20.7% of mosaics (18 out of 87) were CRFs-like isolates, mostly represented by CRF28/CRF29_BF-like viruses (14 out of 18). We also identified five highly supported clusters that together comprise 42 out of 87 (48.3%) BF1 sequences, each cluster containing at least five sequences sharing a similar mosaic structure, suggesting possible new unidentified CRFs_BF1. The divergence time of these five potential new CRFs_BF1 clusters was estimated using a Bayesian approach and indicate that they probably originated between the middle 1980s and the middle 1990s. DNA was extracted from whole blood and four overlapping fragments were amplified by PCR providing full/near full length genomes (FLG/NFLG) and partial genomes. Eleven HIV-1 isolates from Cluster # 5 identified in epidemiologically unlinked individuals living in Central West and North regions provided FLG/NFLG/partial genome sequences with identical mosaic structure. These viruses differ from any known CRF_BF1 reported to date and were named CRF90_BF1 by the Los Alamos National Laboratory. This is the 9th CRF_BF1 described in Brazil and the first one identified in Central West and North regions. Our results highlight the importance of continued molecular screening and surveillance studies, especially of full genome sequences to understand the evolutionary dynamics of the HIV-1 epidemic in a country of continental dimensions as Brazil.

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Section: Introductionmentioning

confidence: 98%

Characterization of HIV-1 CRF90_BF1 and putative novel CRFs_BF1 in Central West, North and Northeast Brazilian regions

Reis¹,

Bello

Guimarães

et al. 2017

PLoS ONE

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“…In order to simulate the application of this gene-editing approach on a diverse within-host virus population, we used a published dataset of HIV sequences obtained from HIV-infected blood donors in Brazil [25], focusing on the pol gene (because it is the most highly conserved) for 10 patients. We started with our list of all pol target sites that we identified above from group and subtype consensus sequences from 2016 LANL alignments, labelling each target site according to the consensus sequence it was identified from (300, 317, 304 and 328 target sites from group M and subtype A-C consensus sequences respectively, 1249 sites total, 897 unique sites).…”

Section: Resultsmentioning

confidence: 99%

“…We started with our list of all pol target sites that we identified above from group and subtype consensus sequences from 2016 LANL alignments, labelling each target site according to the consensus sequence it was identified from (300, 317, 304 and 328 target sites from group M and subtype A-C consensus sequences respectively, 1249 sites total, 897 unique sites). From this combined list of globally conserved target sites, we determined whether each site was present in each patient’s HIV consensus sequence (Tables S4 and S5) [25]. Across infected persons, an average of 89.4 group M target sites (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…To this end, we identified gRNA target sites in HIV LTR that were highly conserved in global consensus sequences and tested the activity of these guides in vitro . Using a separate set of deep-sequence data [25], we showed that sites identified from our list of globally conserved targets that were present in the patient’s sequence also showed greater within-host conservation.…”

Section: Discussionmentioning

confidence: 99%

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Viral diversity is an obligate consideration in CRISPR/Cas9 designs for HIV cure

Roychoudhury

Reeves

et al. 2018

Preprint

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17RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV 18 genome from latently infected cells and have therefore been proposed as a curative approach 19 for HIV. However, most studies to date have focused on molecular clones with ideal target site 20 recognition and do not account for target site variability observed within and between patients. 21For clinical success and broad applicability, guide RNA (gRNA) selection must account for 22 circulating strain diversity and incorporate the within-host diversity of HIV. To address this, we 23 identified a set of gRNAs targeting HIV LTR, gag and pol using publicly available sequences for 24 these genes. We ranked gRNAs according to global conservation across HIV-1 group M and 25 within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet 26 sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all 27 globally-available sequences. We then selected 59 gRNAs from our list of highly-conserved LTR 28 target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We 29 achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active 30 gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-31 infected patients, we found that globally conserved sites also had greater within-host target 32 conservation. Lastly, we developed a mathematical model based on varying distributions of 33 within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the 34 number of doses required to deplete the latent reservoir and achieve functional cure thresholds. 35Our modeling results highlight the importance of within-host target site conservation. While 36 increased doses may overcome low target cleavage efficiency, inadequate targeting of rare 37 strains is predicted to lead to rebound upon ART cessation even with many doses. 38 39 40 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/255133 doi: bioRxiv preprint first posted online Jan. 27, 2018; Author summary 41The field of genome engineering has exploded over the last decade with the discovery of 42 targeted endonucleases such as CRISPR/Cas9. Endonucleases are now being used to develop 43 a wide range of therapeutics and their use has expanded into antiviral therapy against latent 44 viral infections like HIV. The idea is to induce mutations in latent viral genomes that will render 45 them replication-incompetent, thereby producing a functional cure. Although a great deal of 46 progress has been made, most studies to date have relied on molecular clones that represent 47 "ideal" targets. For clinical success and broad applicability, these therapies need to account for 48 viral genetic diversity within and between...

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“…nucleic acid extraction, reverse transcription, PCR, template amplicon preparation for NGS and NGS sequencing) and NGS data processing . The gross error rates generated from short‐read NGS platforms ranges from approximately 1 to 10 errors per 1000 bases leading to increased false positive detection of minority variants when their prevalence falls below approximately 1% . The additional variant QC strategies significantly improve the reliability of calling variants of low abundance, undetectable by Sanger sequencing.…”

Section: Discussionmentioning

confidence: 99%

Bioinformatic data processing pipelines in support of next‐generation sequencing‐based HIV drug resistance testing: the Winnipeg Consensus

Enns

Brumme

et al. 2018

J Intern AIDS Soc

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IntroductionNext‐generation sequencing (NGS) has several advantages over conventional Sanger sequencing for HIV drug resistance (HIVDR) genotyping, including detection and quantitation of low‐abundance variants bearing drug resistance mutations (DRMs). However, the high HIV genomic diversity, unprecedented large volume of data, complexity of analysis and potential for error pose significant challenges for data processing. Several NGS analysis pipelines have been developed and used in HIVDR research; however, the absence of uniformity in data processing strategies results in lack of consistency and comparability of outputs from different pipelines. To fill this gap, an international symposium on bioinformatic strategies for NGS‐based HIVDR testing was held in February 2018 in Winnipeg, Canada, convening laboratory scientists, bioinformaticians and clinicians involved in four recently developed, publicly available NGS HIVDR pipelines. The goal of this symposium was to establish a consensus on effective bioinformatic strategies for NGS data management and its use for HIVDR reporting.DiscussionEssential functionalities of an NGS HIVDR pipeline were divided into five analytic blocks: (1) NGS read quality control (QC)/quality assurance (QA); (2) NGS read alignment and reference mapping; (3) HIV variant calling and variant QC; (4) NGS HIVDR reporting; and (5) extended data applications and additional considerations for data management. The consensuses reached among the participants on all major aspects of these blocks are summarized here. They encompass not only recommended data management and analysis strategies, but also detailed bioinformatic approaches that help ensure accuracy of the derived HIVDR analysis outputs for both research and potential clinical use.ConclusionsWhile NGS is being adopted more broadly in HIVDR testing laboratories, data processing is often a bottleneck hindering its generalized application. The proposed standardization of NGS read QC/QA, read alignment and reference mapping, variant calling and QC, HIVDR reporting and relevant data management strategies in this “Winnipeg Consensus” may serve as a starting guideline for NGS HIVDR data processing that informs the refinement of existing pipelines and those yet to be developed. Moreover, the bioinformatic strategies presented here may apply more broadly to NGS data analysis of microbes harbouring significant genomic diversity.

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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

Cited by 28 publications

References 38 publications

Characterization of HIV-1 CRF90_BF1 and putative novel CRFs_BF1 in Central West, North and Northeast Brazilian regions

Characterization of HIV-1 CRF90_BF1 and putative novel CRFs_BF1 in Central West, North and Northeast Brazilian regions

Viral diversity is an obligate consideration in CRISPR/Cas9 designs for HIV cure

Bioinformatic data processing pipelines in support of next‐generation sequencing‐based HIV drug resistance testing: the Winnipeg Consensus

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