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

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.

Fig 1. Summary of the subtype distribution of viruses from the 257 long-standing HIV-1 infected blood donors analyzed.

The number of viruses that belong to each subtype is indicated in the relevant bar chart section. The regions of origin are color-coded and indicated by arrows.

Fig 2. Phylogenetic tree constructed using a ML method from the non-recombinant NFLG sequences of 149 blood donor samples, 53 non-redundant B, eight subclade F1, and eight subtype C sequences from Brazil retrieved from GenBank database.

Besides these sequences, the tree also included 37 reference strains (Los Alamos database) representing subtypes A-D, F-H, J and K. Sequences identified in this study are indicated by circles and the geographic origin of each sequence is color-coded. Monophyletic clusters from the same geographic origin are indicated by thick green branches. For clarity purposes, the tree was midpoint rooted. Gray arrows indicate hypermutated sequences with a predominance of unidirectional G-to-A mutations (010BR_MG005, and 010BR_SP022). The aLRT values of at least 80% are indicated at nodes. The scale bar represents 0.05 nucleotide substitutions per site.

Fig 3. 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.

Fig 4. Phylogenetic trees were constructed using a maximum-likelihood method from partial pol plasma RNA virus (1182 bp), concatenated regions of subtype B sequences (provirus nt 1724–1980, 3284–3498) and proviral DNA (4978 bp fragment) from patient 010BR_RJ033 along with 53 non-redundant Brazilian subtype B and HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

The recombinant proviral fragment (2173 bp) is evidenced by a Bootscan plot. Average coverage reads of both proviral fragments are indicated.