Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus Type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (H IV-I) gag subtypes was developed based on restriction fragment length polym orphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PC R) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650 bp) regions. The consensus sequences of subtypes A-D, the only subtyp es identified in South Africa, were analyzed to detect restriction endonucl eases which generate unique patterns for each subtype. Four restriction end onucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 b p fragment with AluI allowed identification of subtype C. Samples not ident ified were then reamplified, and a 650 bp fragment digested with AccI to id entify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by ei ther sequence analysis of the gag p17 region (n = 33); or heteroduplex mobi lity assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of t he 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 5 6 as subtype C and one as a subtype D virus. No discrepancies were found be tween RFLP gag subtypes and gag sequence subtypes demonstrating the reliabi lity of this method. There was also no discordance between gag RFLP subtype s and env HMA subtypes, suggesting that there were no recombinant viruses d etected relating to the genomic regions analyzed. RFLP is an effective tech nique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa. (C) 1999 Elsevier Science B.V. All rights reserved.