Title: Long-range HIV genotyping using viral RNA and proviral DNA for analysis of HIV drug-resistance and HIV clustering
Authors: Novitsky V, Zahralban-Steele M, McLane MF, Moyo S, Widenfelt E, Gaseitsiwe S, Makhema J, Essex M.
Journal: Journal of Clinical Microbiology,in press:in press (2015)
The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (J Clin Microbiol 50:3838?44, 2012), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIVinfected subjects with suppressed viral load (e.g., subjects on ART).
The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (NRTIs and NNRTIs), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and number of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies.
The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral load, including 65% of patients with undetectable levels of HIV-1 RNA load. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultra-deep sequencing. The developed method is cost-effective ? the cost of the long-range.
Citation: Novitsky V, Zahralban-Steele M, McLane MF, Moyo S, Widenfelt E, Gaseitsiwe S, Makhema J, Essex M. Long-range HIV genotyping using viral RNA and proviral DNA for analysis of HIV drug-resistance and HIV clustering Journal of Clinical Microbiology,in press:in press (2015).