Salazar-Gonzalez Jesus F, Bailes Elizabeth, Pham Kimmy T, Salazar Maria G, Guffey M Brad, Keele Brandon F, Derdeyn Cynthia A, Farmer Paul, Hunter Eric, Allen Susan, Manigart Olivier, Mulenga Joseph, Anderson Jeffrey A, Swanstrom Ronald, Haynes Barton F, Athreya Gayathri S, Korber Bette T M, Sharp Paul M, Shaw George M, Hahn Beatrice H
Department of Medicine, University of Alabama at Birmingham, 720 20th Street South, Kaul 816, Birmingham, AL 35294, USA.
J Virol. 2008 Apr;82(8):3952-70. doi: 10.1128/JVI.02660-07. Epub 2008 Feb 6.
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 nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.
准确识别传播的病毒及其衍生的序列，对于阐明1型人类免疫缺陷病毒（HIV-1）的传播以及开发预防感染的疫苗、药物或杀菌剂可能具有重要作用。在此，我们描述了一种实验方法，即将HIV-1 env基因作为完整的遗传单位进行分析，这些基因通过单基因组扩增（SGA）从血浆病毒粒子RNA中扩增得到，随后对未克隆的DNA扩增子进行直接测序。我们表明，该策略可避免由Taq诱导的核苷酸替换和模板切换所导致的体外假象，能准确呈现体内env准种的情况，并且总体错误率（包括核苷酸错配、插入和缺失）小于8×10⁻⁵。将此方法应用于对12名赞比亚受试者血浆中的病毒进行分析，这些受试者在血清转化后3个月内采集了样本，我们发现可以识别出传播的或早期的奠基病毒，并且能够确定早期env多样化的分子途径和速率。具体而言，我们发现12名受试者中有8名各自感染了单一病毒，而另外4名感染了不止一种病毒；在一名受试者血清转化后的80天内，病毒进化速率为每天每个位点1.7×10⁻⁵个替换；并且基于非同义突变的非随机积累，在Env和重叠的Rev序列中可以看到强烈免疫选择的证据。我们还将SGA方法的结果与对相同患者样本进行的更传统的批量PCR扩增方法的结果进行了比较，发现后者与Taq诱导的重组、核苷酸错配、模板重采样和克隆偏差的过高发生率相关。这些发现表明，通过对大规模疫苗试验中典型间隔采集的血浆病毒进行SGA分析，可以识别出导致临床感染的HIV-1 env基因、其他病毒基因甚至全长病毒基因组，并且能够准确确定病毒多样化和免疫逃逸的途径。
