HIV-1 subtyping using phylogenetic analysis of pol gene sequences

doi:10.1016/S0166-0934(01)00272-5

Journal of Virological Methods

Volume 94, Issues 1–2, May 2001, Pages 45-54

https://doi.org/10.1016/S0166-0934(01)00272-5 Get rights and content

Abstract

HIV-1 pol gene sequencing is now used routinely in France to identify mutations associated with resistance to reverse transcriptase (RT) or protease (PR) inhibitors. These sequences may also provide other information, such as the HIV-1 subtype. HIV-1 subtyping was compared using the RT and PR gene sequences to heteroduplex mobility assay (HMA) of the envelope gene. The RT and PR genes of 51 samples that had been subtyped earlier by HMA were sequenced. Sequences were aligned and subtypes were determined by phylogenetic analysis with reference HIV sequences. HMA gave the following subtypes: A (20), B (19), C (1), D (3), F (1), G (3) and CRF01-AE (4). Phylogenetic analysis of the RT gene gave: A (5), B (19), C (2), D (3), F (1), G (6), J (2), CRF01_AE (4), CFR02_AG (7) and undetermined (2). PR gene analysis did not infer subtypes with sufficient confidence. HMA and RT subtyping was not in agreement in nine cases. RT subtyping can identify CFR02_AG and CRF01_AE variants from A subtype RT. It was shown that phylogenetic analysis of the RT gene could provide a useful method for HIV-1 subtyping. The length of the amplicon and the relative performance of each primer pair used in this study favoured RT sequences as a subtyping tool. One potential advantage over env subtyping HMA is the ability to identify some circulating recombinant forms (CRFs).

Introduction

HIV-1 is known for its remarkable genetic variability. HIV-1 variants are randomly generated during virus replication and then selected by the host environment. Two mechanisms are responsible for producing virus variants. One is the error-prone nature of the reverse transcriptase (RT), which has no proofreading function and causes nucleotide substitutions, deletions and insertions (Preston and Dougherty, 1996, Mansky, 1998). The second is the recombination generated by the low processivity of RT and the presence of two copies of genomic RNA in the nucleocapsid (Quinones-Mateu and Arts, 1999). Both mechanisms intervene during HIV-1 replication, which occurs at high rate (10¹⁰ viral particles produced each day) and continues for long periods within individuals and human populations (Ho et al., 1995, Perelson et al., 1997). The newly produced variants survive only if they can compete with other variants to replicate and escape immune responses and antiretroviral molecules. The fittest variant for a specific micro environment is then selected and may become a major variant.

HIV-1 variants has been divided into groups (M, N and O) and subtypes; A, B, C, D, F1, F2, G, H, J, K for group M based on phylogenetic analyses of complete genome nucleotide sequences (Robertson et al., 2000). Intersubtype recombinant viruses have been identified and recently named as CRF01 to CRF06 (circulating recombinant forms (CRFs)) (Robertson et al., 2000). HIV-1 group M viruses are distributed world-wide and are the most common in developed countries. The distribution of group subtypes is both geographical and epidemiological. HIV-1 subtypes are relevant epidemiological tools for studying the evolution of the epidemic. In those studies, HIV-1 subtypes are often determined using V3 serotyping or env heteroduplex mobility assays (Arens, 1999).

The env sequences are used preferentially for subtyping because of their great variability. Nucleotide sequences from other HIV-1 genes that also vary significantly can also be useful for subtyping HIV-1 and can be of value for identifying recombinant genomes. The development of RT and protease (PR) gene sequencing for searching for mutations conferring resistance to RT and PR-inhibitors has made nucleotide sequences from those regions available as an alternative method for subtyping HIV-1.

Subtyping of HIV-1 was compared using phylogenetic analysis of RT and PR nucleotide sequences with that using a heteroduplex mobility assay on env sequences, which is a method used widely. Samples giving discordant results were also analysed by sequencing the env region.

Section snippets

Samples

A total of 51 HIV-1 strains from patients living in the Toulouse area were used. These strains had been subtyped using the heteroduplex mobility assay (HMA) to identify non-B subtype HIV-1 infections in several settings. First, they were used for patients (n=21) with differences in plasma HIV-1 RNA concentration by bDNA and RT-PCR Monitor assays, or by Monitor v1.0 and v1.5 assays (Roche Diagnostic, Meylan France). Second, they were used for patients (n=18) with a low CD4 cell count and low and

HMA subtyping

HIV-1 isolates were subtyped by heteroduplex mobility assay using a 600 bp DNA fragment overlapping the V3 region. As indicated in Table 1, HIV-1 subtypes were B (n=19), A (n=20), C (n=1), D (n=3), F (n=1), G (n=3) and CRF01-AE (n=4).

RT gene subtyping

A 774 bp from RT gene was amplified, sequenced directly and subtyped by phylogenetic analysis using reference sequences and the Neighbor-Joining method (Fig. 1). The RT gene was amplified in all cases. The HIV-1 subtype was determined in 47/51 (92%) strains.

Discussion

The use of HIV-1 RT and PR genotyping for determining resistance mutations will produce a large amount of pol gene sequence data. These available sequences may provide complementary information to physicians, particularly for HIV-1 subtypes. This study assesses the performance of HIV-1 subtyping using RT and PR sequences for phylogenetic analysis, using HMA, a technique, which is used widely, as reference.

The set of strains used in this study is not representative of the distribution of HIV-1

Acknowledgements

We thank Dr Owen Parkes and Monica Ghosh for linguistic advice.

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HIV subtype C and C-related recombinants have remained the major circulating genotypes in Bangladesh. Although the recurring transmission of subtype C occurred among PWID, we identified possible transmission to other key populations (KPs), which suggests spillover from PWID through the sexual route. The prevalence of drug-resistant mutation was low, and all strains were susceptible to NRTIs and NNRTIs drugs. Unique recombination forms (URF) with genotype C for gag–pol and A1 for env was also identified.
The study findings warrant continuous monitoring of HIV-positive individuals and future investigation to identify social networks within and between KPs to halt the transmission and prevent new infections.
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The gold-standard comprehensive classification of HIV-1 subtypes is based on phylogenetic analyses of whole genomes (Robertson et al., 2000), despite their rare availability. Conversely, HIV pol sequences, collected as routine clinical care to assess for drug resistance mutations, have enough phylogenetic signal to accurately determine the pol subtype (Hué et al., 2004; Pasquier et al., 2001; Snoeck et al., 2002; Yahi et al., 2001). Other subtyping methods include commonly used algorithms such as the Stanford HIV Database (Liu and Shafer, 2006), the REGA HIV subtyping tool (de Oliveira et al., 2005), Los Alamos Recombinant Identification Program (RIP) (Siepel et al., 1995), the National Center for Biotechnology Information (NCBI) tool (Rozanov et al., 2004), Geno2Pheno (Beerenwinkel et al., 2002), EuResist (Rosen-Zvi et al., 2008), Jumping Profile Hidden Markov Model (jpHMM) (Schultz et al., 2006), and Subtype Analyzer (STAR) (Gale et al., 2004).
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Such method has been proven useful for subtyping (Gouy et al., 2010; Posada and Crandall, 2001), and it saves computation time. A query sequence was assigned to a particular clade if it clustered monophyletically inside that clade with bootstrap support >70% (Hillis and Bull, 1993; Pasquier et al., 2001; Yahi et al., 2001). Otherwise, the query sequence was considered discordant.
To investigate differences in pathogenesis, diagnosis and resistance pathways between HIV-1 subtypes, an accurate subtyping tool for large datasets is needed. We aimed to evaluate the performance of automated subtyping tools to classify the different subtypes and circulating recombinant forms using pol, the most sequenced region in clinical practice. We also present the upgraded version 3 of the Rega HIV subtyping tool (REGAv3).
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Figure 1 shows the increasing percentage of patients in care with non-B subtype infections over the last decade. As viral diversity expands, the traditional viral subtype classifications may need to be combined with the standardized resistance genotype assignment database.32,52 Much more research also needs to be done to determine the optimal laboratory support and the differences in disease course, response to treatment, and resistance patterns that emerge under selective drug pressure for all non-B subtype infections.53,54
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Journal of Virological Methods

Abstract

Introduction

Section snippets

Samples

HMA subtyping

RT gene subtyping

Discussion

Acknowledgements

References (19)

Recovery of virtually full-length HIV-1 provirus of diverse subtypes from primary virus cultures using the polymerase chain reaction

Virology

Methods for subtyping and molecular comparison of human viral genomes

Clin. Microbiol. Rev.

Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes

J. Virol.

Genetic subtyping of human immunodeficiency virus using a heteroduplex mobility assay

Pcr. Methods Appl.

Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes

Science

A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1

J. Virol.

Simplified strategy for detection of recombinant human immunodeficiency virus type 1 group M isolates by gag/env heteroduplex mobility assay

J. Virol.

Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection

Nature

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Automated subtyping of HIV-1 genetic sequences for clinical and surveillance purposes: Performance evaluation of the new REGA version 3 and seven other tools

Evaluation of the QIAsymphony™ SP and Artus™ RealTime extraction-quantification systems for measuring HIV-1 virus load

HIV-1 viral diversity and its implications for viral load testing: Review of current platforms