Biological Properties of HIV-1 V3 Evolutionary Variants

项目总结/摘要 病毒基因序列代表了关于生物过程的丰富和有价值的信息来源。 在过去的十年中，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， 克服了传统NGS的几个严重限制，包括大大减少了错误掺入， - 通过先前的PCR步骤引入的重组，减少测序平台的错误，和 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 集在本申请中，我们将介绍如何将Primer ID与NGS一起使用，以解决 在无治疗、治疗失败和治疗失败的情况下病毒演变背景下的HIV-1人群分析， 进行抑制性治疗此外，我们将把我们的序列数据与最先进的进化分析联系起来 其目标是使高质量的数据集和工具易于用于进一步的二次分析。在目标1中， 我们将获得28名HIV感染妇女（来自WIHS队列）的纵向血浆样本， CD 4 + T细胞计数，直至CD 4 + T细胞计数低于100个细胞/µL。我们将执行 多重引物ID测序以获得接近全长的HIV-1基因组。我们预测，X4变异体在病毒 种群首先出现在低丰度，我们将能够检测到比以前观察到的要早得多。 此外，我们将调查所有测序区域的纵向病毒多样性变化，以评估人群 动态并将所有这些标志物与炎症标志物、CNS损伤以及 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 用于耐药性测试的筛选工具。我们假设，耐药突变的可能性， 介导的逃逸可能发生在微小的变异体上，微小的变异体太小而不能用具有 被用于约会。我们将分析来自8号医院HIV诊所的大型受试者队列的样本。 中国广州的一家医院，以确定通过评估治疗失败而遗漏微小变异的频率 使用桑格测序，确定传播或预先存在的突变在WHO失败中的作用 线治疗，并确定突变模式后，随后失败的WHO二线治疗。在目标3中， 我们将使用Primer ID深度测序来监测接受治疗的受试者的群体变化。我们 假设即使在治疗期间，低水平病毒群体也会发生变化， 使用深度监测。所获得的见解将为如何应用这项技术搜索 与X4病毒相关的进化变异，以确定变化的病毒群体的其他生物学相关性 与女性疾病进展相关，以确定病毒群体是如何建立的， 次要序列变异的稳态水平，以检查耐药变异在治疗中的作用 失败，并探讨NGS技术在治疗受试者中的效用。

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.