Development and Application of a High-Throughput HIV Type 1 Genotyping Assay to Identify CRF02_AG in West/West Central Africa

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Abstract
In West/West Central Africa, CRF02_AG is the most prevalent HIV-1 strain and circulates in the milieu of rare subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs). The molecular complexity of HIV-1 epidemics in this region and the need to extensively sample large populations, such as in the case of vaccine trials, pose seemingly conflicting requirements between full-genome sequencing and high-throughput low-resolution assays. Here we describe the development and evaluation of a multiregion hybridization assay (MHAcrf02) for the efficient genotyping of CRF02_AG in West/West Central Africa. Subtype A, G, and CRF02_AG-specific fluorescent probes were designed flanking five recombination breakpoints in CRF02_AG and were used in real-time PCRs. A panel representing West/West Central African HIV-1 genetic diversity was evaluated by MHAcrf02. The sample set, previously characterized by full-genome sequencing, included CRF02_AG and CRF02_AG-containing recombinants (n = 28), other subtypes, CRFs, and URFs (n = 34). DNA from peripheral blood mononuclear cells, cocultures, and plasmids was used as template. When the patterns of probe reactivity were evaluated. CRF02_AG was identified with a 100% specificity and sensitivity. In conclusion, MHAcrf02 will permit more efficient characterization of HIV-1 in West/West Central Africa, where CRF02_AG is an important strain. Together with other regional genotyping assays MHAcrf02 will contribute to the development of a global picture of HIV-1 diversity and geographic distribution, providing a strong foundation for intervention, including vaccine development.

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