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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes
Journal of Virological Methods, Volume 136, No. 1-2, Year 2006
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Description
Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization. © 2006 Elsevier B.V. All rights reserved.
Authors & Co-Authors
Rousseau, Christine M.
United States, Seattle
University of Washington
Birditt, Brian A.
United States, Seattle
University of Washington
McKay, Angela R.
United States, Seattle
University of Washington
Stoddard, Julia N.
United States, Seattle
University of Washington
Lee, Tsan Chun
United States, Seattle
University of Washington
McLaughlin, Sherry
United States, Seattle
University of Washington
Moore, Sarah W.
United States, Seattle
University of Washington
Shindo, Nice
United States, Seattle
University of Washington
Learn, Gerald H.
United States, Seattle
University of Washington
Korber, Bette T.
United States, Los Alamos
Los Alamos National Laboratory
Brander, Christian
United States, Boston
Massachusetts General Hospital
Goulder, Philip Jeremy Renshaw
United States, Boston
Massachusetts General Hospital
United Kingdom, Oxford
Nuffield Department of Medicine
Kiepiela, Photini
South Africa, Durban
University of Kwazulu-natal
Walker, Bruce D.
United States, Boston
Massachusetts General Hospital
United States, Chevy Chase
Howard Hughes Medical Institute
Mullins, James I.
United States, Seattle
University of Washington
Statistics
Citations: 104
Authors: 15
Affiliations: 6
Identifiers
Doi:
10.1016/j.jviromet.2006.04.009
ISSN:
01660934
Research Areas
Infectious Diseases
Study Design
Cross Sectional Study
Cohort Study
Study Locations
South Africa