Surveillance technology for HIV-1 subtype C in Ethiopia: An env-based NASBA molecular beacon assay to discriminate between subcluster C and C′
Introduction
Global tracking of the genetic variability of human immunodeficiency virus-type 1 is important from both epidemiological and treatment perspectives. The identification of HIV-1 clades or subtypes (Robertson et al., 2000) relies on sequence analysis of the env, gag or pol genes (Myers et al., 1994, Pasquier et al., 2001, Njouom et al., 2003). Sequencing remains the gold standard but other subtyping technologies that are less costly, offering ease of performance and which are therefore more practical to adopt for large population-based screening in developing countries have undergone extensive evaluation (Barin et al., 1996, Luigi et al., 1995, Luo et al., 1998).
HIV-1 subtype C is estimated to account for at least 56% of all global infections (Esparza and Bhamarapravati, 2000). The HIV-1 epidemic in Ethiopia is overwhelmingly subtype C, since the start of the epidemic over two decades ago (Abebe et al., 2001a, Abebe et al., 2001b). Sequence analysis has demonstrated the presence of a distinct subcluster (C prime or C′) within the main subtype C group viruses in Ethiopia (Abebe et al., 2000), which is increasing in prevalence over time in surveyed population groups. The functional relevance of two distinct genetic subclusters of HIV-1 subtype C in Ethiopia remains to be investigated. The development of a rapid, sensitive and highly specific assay to discriminate between the two genetic subclusters of HIV-1 subtype C circulating in Ethiopia based on the gag gene has previously been reported (Ayele et al., 2004). In the present study, the development of an env (C2V3) gene-based assay utilizing a similar real-time NASBA format to discriminate between subcluster C and C′ is described. Genotyping of C2V3 is of interest since there is some indication that a C′ envelope gene may confer a selective transmission advantage as compared to C (Pollakis et al., 2003). This is inferred from the observation that among recombinant isolates C′ predominates in the envelope gene, and therefore the continued evaluation of the C/C′ ratio would be a good indicator of the evolution dynamics in the two regions of the genome.
The two regions (gag and env) were targeted for development of assays to discriminate between the subclusters as these contain the principal immunodominant regions which are important to consider in vaccine design (Van Baalen et al., 1996, Neurath and Strick, 1990). The env-based assay in combination with the gag-based test is expected to aid in surveillance efforts that would in addition, greatly aid in identifying functional differences between C and C′ viruses in Ethiopia. Application of this tool will enable detailed analysis of differences, if any, in disease progression patterns over the long term in individuals infected with one or the other subcluster, which might be ascribed to the intrinsic biological properties of those viruses.
Section snippets
Sample selection
Plasma samples from 49 HIV-seropositive individuals enrolled in the Ethio-Netherlands AIDS Research Project (ENARP) cohort to study the natural history of HIV infection in Ethiopia were selected. Details of the cohort are as described elsewhere (Sahlu et al., 1998).
HIV-1 extraction and amplification
HIV-1 RNA was isolated from 200 μl each of plasma using the silica-based Boom extraction technique for nucleic acids (Boom et al., 1990). Viral load was determined for all individuals using the NucliSens HIV-1 QT assay (bioMérieux,
Results
During assay development various beacon combinations from both loci were tested and genotyping results coming closest to those generated by DNA sequencing were found using the two C beacons (MB146 and MB148) in combination with one C′ beacon located in the downstream region of the PCR amplified fragment (MB147) (data not shown). Hence, this triple beacon combination (MB146/147/148) was adopted as the first-line screening assay for genotyping all 49 samples in the present study. The method was
Discussion
This study describes the performance of a novel env-based genotyping assay having a high sensitivity and specificity to distinguish between the two main subclusters of HIV-1 subtype C circulating in Ethiopia. The assay is tailor-made to the Ethiopian situation where HIV-1 subtype C remains fairly homogeneous, indicating perhaps a more recent epidemic compared to places, like South Africa where multiple lineages of HIV-1 subtype C have been reported (Gordon et al., 2003).
Concordance in results
Acknowledgements
This study is a part of the Ethio-Netherlands AIDS Research Project (ENARP), a collaborative effort of the Ethiopian Health and Nutrition Research Institute (EHNRI, Addis Ababa, Ethiopia), the Amsterdam Municipal Health Service (GG/GD), the Academic Medical Center of the University of Amsterdam (AMC), and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB, Amsterdam, the Netherlands). Financial support of ENARP is from the Netherlands Ministry of Foreign Affairs
References (29)
- et al.
Accelerating the development and future availability of HIV-1 vaccines: why, when, where and how?
Lancet
(2000) - et al.
Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the env glycoprotein of the human immunodeficiency virus-type 1 (HIV-1)
Mol. Immunol.
(1990) - et al.
Assessment of HIV-1 subtyping for Cameroon strains using phylogenetic analysis of pol gene sequences
J. Virol. Meth.
(2003) - et al.
HIV-1 subtyping using phylogenetic analysis of pol gene sequences
J. Virol. Meth.
(2001) - et al.
Identification of a genetic subcluster of HIV-1 subtype C (C′) widespread in Ethiopia
AIDS Res. Hum. Retrov.
(2000) - et al.
Timing of the introduction into Ethiopia of the subcluster C′ of HIV-1 subtype C
AIDS Res. Hum. Retrov.
(2001) - et al.
Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent diversification
AIDS
(2001) - et al.
Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus-type 1 subtype C and C′ infections in Ethiopia
J. Clin. Microbiol.
(2004) - et al.
Diversity of antibody binding to V3 peptides representing consensus sequences of HIV-type 1 genotypes A–E: an approach for HIV type 1 serological subtyping
AIDS Res. Hum. Retrov.
(1996) - et al.
Rapid and simple method for purification of nucleic acids
J. Clin. Microbiol.
(1990)
Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus-type 1 isolates from groups M, N and O
J. Clin. Microbiol.
Molecular characterisitics of human immunodeficiency virus-type 1 subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies
J. Virol.
An empirical test for bootstrapping as a method for assessing confidence in phylogenetic analysis
Syst. Biol.
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