Biological Properties of HIV-1 V3 Evolutionary Variants

项目摘要/摘要 病毒基因序列是有关生物过程的丰富而有价值的信息来源。 过去十年下一代测序(NGS)技术的进步现在提供了机会 以前所未有的方式探索病毒种群的多样性。我们最近开发了Primer ID策略，以 克服传统NGS的几个严重限制，包括极大地减少误合并和 通过前面的PCR步骤引入的重组，减少了测序平台的误差，以及 揭示最终数据中实际表示的初始病毒基因组/模板的采样深度 准备好了。在此应用程序中，我们描述了如何结合使用Primer ID和NGS来解决 在缺乏治疗、治疗失败的情况下的病毒进化背景下的艾滋病毒-1种群分析，以及 用抑制疗法治疗。此外，我们将把我们的序列数据与最先进的进化分析联系起来 目标是使高质量的数据集和工具随时可用于进一步的二次分析。在目标1中， 我们将获得28名艾滋病毒感染妇女的纵向血浆样本(来自WIHS队列)，从HIGH开始 直到他们进展到低于100个/微升的CD4+T细胞计数。我们将进行 多重引物ID测序以获得接近全长的HIV-1基因组。我们预测病毒中的X4变异体 种群首先出现在低丰度的时候，我们将能够比之前观察到的更早地发现。 此外，我们将调查所有测序区域的纵向病毒多样性变化，以评估种群 动力学，并将所有这些标志物与炎症、中枢神经系统损伤以及广度和效力的标志物联系起来 中和抗体。在目标2中，我们将应用多重引物ID测序方法作为 耐药性检测的筛查工具。我们假设耐药突变的可能性是 中介转义可能发生在次要变体中，这些变体太小，无法使用具有 一直沿用到现在。我们将分析来自8号艾滋病诊所的大量受试者的样本。 广州一家医院的中国通过评估治疗失败来确定微小变异被遗漏的频率 使用Sanger测序，确定传播的或先前存在的突变在WHO First失败中的作用 一线治疗，并确定世卫组织二线治疗随后失败后的突变模式。在《目标3》中， 我们将使用Primer ID的深度测序来监测治疗对象的群体变化。我们 假设低水平病毒种群的变化即使在治疗期间也会发生，并且这些变化可能是 使用深度监控。所获得的见解将对如何应用这项技术来搜索 与X4病毒相关的进化变异，以确定不断变化的病毒种群的其他生物相关性 随着女性疾病的发展，来定义病毒种群是如何建立的，关于 稳定水平的次要序列变异，以检查耐药变异在治疗中的作用 失败，并探索NGS技术在治疗科目设置中的应用。

Project Summary/Abstract Viral gene sequences represent a rich and valuable source of information about biological processes. Advances in next generation sequencing (NGS) technology over the past decade now provide the opportunity to probe viral population diversity in unprecedented ways. We recently developed the Primer ID strategy to overcome several serious limitations of conventional NGS including greatly reducing the mis-incorporation and recombination introduced by the preceding PCR step, reducing the errors of the sequencing platform, and revealing sampling depth of the initial viral genomes/templates that are actually represented in the final data set. In this application we describe how we will use Primer ID with NGS to address long-standing issues in HIV-1 population analysis in the context of viral evolution in the absence of therapy, with failed therapy, and with suppressive therapy. In addition, we will link our sequence data to state-of-the-art evolutionary analysis with a goal of making high quality data sets and tools readily available for further secondary analyses. In Aim 1, we will obtain longitudinal plasma samples of 28 HIV-infected women (from the WIHS cohort) starting with high CD4+ T cell counts until they progress to CD4+ T cell counts of less than 100 cells/µL. We will perform multiplexed Primer ID sequencing to obtain near full length HIV-1 genome. We predict that X4 variants in viral populations first emerge at low abundance that we will be able to detect much earlier than previously observed. In addition, we will investigate longitudinal viral diversity changes in all sequenced regions to assess population dynamics and link all of these markers to markers of inflammation, CNS damage, and the breadth and potency of neutralizing antibodies. In Aim 2, we will apply the multiplexed Primer ID sequencing approach as a screening tool for drug resistance testing. We hypothesize that the potential for drug resistance mutations to mediate escape can occur from minor variants, too minor to be reliably detected with the methods that have been used to date. We will analyze samples from a large cohort of subjects attending the HIV clinic at the #8 Hospital in Guangzhou, China, to determine how often minor variants are missed by assessing therapy failure using Sanger sequencing, determine the role of transmitted or pre-existing mutations in the failure of WHO first line therapy, and define the mutation pattern after subsequent failure of WHO second line therapy. In Aim 3, we will use deep sequencing with Primer ID to monitor population changes in subjects on therapy. We hypothesize that changes in the low level viral populations occur even while on therapy and that these can be monitored using deep. The insights gained will be informative for how to apply this technology to search for evolutionary variants related to X4 viruses, to define other biological correlates of the changing viral population with disease progression in women, to define how the viral population gets established with regard to the steady-state levels of minor sequence variants, to examine the role of drug resistance variants in therapy failure, and to explore the utility of NGS technology in the setting of subjects on therapy.