Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing

Abstract

Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Phylogenetic and Demographic Characterization of Directed HIV-1 Transmission Using Deep Sequences from High-Risk and General Population Cohorts/Groups in Uganda

“…Our sample comprised study participants who were ART naïve or on therapy with unsuppressed virus, and it is possible that transmissions from individuals who reached viral suppression soon after infection are under-represented in our data. Generation of additional HIV-1 deep sequence data based on sequencing protocols that amplify proviral HIV-1 DNA or sequence virus from low viraemic specimens [54,55] is a consideration for our future analyses. Fifth, transmissions including individuals with co-infections may have been excluded by the definition of source-recipient pairs.…”

Validation of Variant Assembly Using HAPHPIPE with Next-Generation Sequence Data from Viruses

“…Finally, we discuss the analyses completed for the pipelines and consensus sequences, along with true and reference sequences used ( Figure 1 ). We compared HAPHPIPE to two other software programs, Geneious and HyDRA, based on their frequent use in viral studies [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 38 , 51 , 52 , 53 , 56 , 57 ], particularly among clinicians and those new to bioinformatics analysis. In particular, we chose HyDRA over similar web-based platforms such as PASeq [ 66 ] due to its popularity in the HIV research community; we chose Geneious as a representative of commercial software frequently used in genomics analysis.…”

“…Many studies implement reference-based assembly [ 10 , 13 , 14 ] with tools such as CLC Main Workbench (Qiagen, Hilden, Germany) [ 15 , 16 , 17 , 18 ], Geneious ( ) [ 19 , 20 , 21 , 22 , 23 , 24 ], HyDRA [ 25 , 26 , 27 , 28 ], SmartGene (Switzerland) [ 21 , 29 , 30 ], PAseq [ 31 , 32 ] and Amplicon Variant Analyzer (AVA; pyrosequencing-based platform) [ 21 , 33 , 34 , 35 , 36 , 37 ]. Other studies complete de novo assembly with tools such as Geneious [ 38 ], CLC Main Workbench (Qiagen) [ 16 , 39 , 40 ], and Iterative Virus Assembler (IVA) [ 22 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. HCV studies follow similar patterns to those of HIV-1 [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ].…”

Are We Ready for NGS HIV Drug Resistance Testing? The Second “Winnipeg Consensus” Symposium

“…Additionally, in high-throughput environments where sample batching is feasible, NGS offers improved time efficiency and cost-effectiveness [37]. Meanwhile, although separate SS assays are required to cover the PR + RT and IN gene when needed, sequencing all three genes simultaneously or even beyond can be easily achieved by using longer PCR amplicons or combining different amplicons prior to the fragmentation and tagmentation steps during NGS library preparation [38,39]. While the large number of clonal NGS reads from a single specimen can enable high-resolution analyses of literally all HIVDR variants, the consensus sequences derived from NGS can also be used to mimic SS output in any downstream applications, such as phylogenetic analysis for molecular epidemiology that often requires "one sample, one sequence".…”