DIRECT SEQUENCING OF HIV-1 gp120 V3 LOOP BY POLIMERASE CHAIN REACTION IN ARGENTINEAN SAMPLES

GOMEZ CARRILLO, M; HODARA V; PICCARDO, C; CALELLO, M; PAMPURO, S; MARTINEZ P L; LIBONATTI O

Amsterdam, Holanda

Conferencia; VIII INTERNATIONAL CONFERENCE ON AIDS; 1992

International AIDS Society

Objective: to perform direct sequencing of HIV-1 gp120 V3 loop using Polimerase Chain Reactin technology in order to compare results obtained in Argentinean samples with HIV-1 strains of other geographical regions. Methods: A total of 20 HIV-1 infected IVDA from Argentina were enrolled in this study after givin their informed consent. All individuals, though asymptomatic, were seropositive for HIV-1 antibodies. PBMCs  were isolated by Ficoll-Hypaque density gradient and cell were lysed. DNA asymetric PCR amplification was performed on lysates using primers from BH10 isolate env gene (7191-7210 and 7532-7513). Amplification was allowed for 24 cycles in a Perkin Elmer Thermal cycler. Reactions were monitored for production of single stranded DNA on 1,5% agarose gel. Sequencing was performed with Sequenase kit and 35 S alpha dNTP and reactions were resolved by electrophoresis in a 6%  polyacrilamide - Urea gel. Results: Percentage of amino acid homology between the obtained consensus sequece and different HIV-1 strains was: SF2 (84%); BH10,SC, HAN (76%); MN (70%); WHJ2 (65%) JH3 (62%). Conclusions: As HIV-1 gp120 V3 loop is a major target in AIDS vaccine attemps its variability may have geographical implications.This study allowed us to know Argentinean V3 loop sequences by direct sequencing methodology avoiding viral culture and subcloning.