Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single-Genome Amplification and Sequencing

作者：Jesus F. Salazar-Gonzalez, Elizabeth Bailes, Kimmy T. Pham, Maria G. Salazar, M. Brad Guffey, Brandon F. Keele, Cynthia A. Derdeyn, PAUL K. FARMER, Eric Hunter, Susan Allen, Olivier Manigart, Joseph Mulenga, Jeffrey A. Anderson, Ronald Swanstrom, Barton F. Haynes, Gayathri S. Athreya, Bette Korber, Paul M. Sharp, George M. Shaw, Beatrice H. Hahn · 发表于：Journal of Virology · 年份：2008 · DOI：10.1128/jvi.02660-07 · 被引用次数：605 · 研究领域：HIV Research and Treatment、Bacteriophages and microbial interactions、Herpesvirus Infections and Treatments

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of ...