Williams Dawnnica, Overbaugh Julie
Division of Human Biology, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA, USA.
J Med Primatol. 2004 Oct;33(5-6):227-35. doi: 10.1111/j.1600-0684.2004.00073.x.
The generation of a diverse population of viral variants is a hallmark of simian immunodeficiency virus (SIV) infection. In order to address what role this diversity plays in disease progression, accurate sampling of the viral population is necessary. However, traditional PCR-based methods often rely on amplification of multiple genomes in one reaction, leading to resampling of viral genomes and potential errors in the estimations of viral diversity, especially when sequences from only one or a small number of PCRs are examined and/or viral copy number is low. Here we describe a method to amplify one viral envelope gene per PCR, thereby avoiding resampling. For this purpose we developed a highly accurate real-time PCR method to quantify SIV copy number, then used a single SIV template in a sensitive, high-fidelity full-length envelope PCR. Using this method, we have estimated the intra-animal viral diversity for a cohort of five pig-tailed macaques (Macaca nemestrina) infected with SIVMne variants, which displayed a broad range of viral loads at setpoint.
产生多种病毒变体是猴免疫缺陷病毒（SIV）感染的一个标志。为了探究这种多样性在疾病进展中所起的作用，对病毒群体进行准确采样是必要的。然而，传统的基于PCR的方法通常依赖于在一个反应中扩增多个基因组，导致病毒基因组的重新采样以及病毒多样性估计中的潜在误差，特别是当仅检查一个或少数几个PCR的序列和/或病毒拷贝数较低时。在这里，我们描述了一种每个PCR扩增一个病毒包膜基因的方法，从而避免重新采样。为此，我们开发了一种高度准确的实时PCR方法来定量SIV拷贝数，然后在灵敏、高保真的全长包膜PCR中使用单个SIV模板。使用这种方法，我们估计了一组五只感染SIVMne变体的猪尾猕猴（食蟹猴）的动物体内病毒多样性，这些猕猴在设定点显示出广泛的病毒载量。
