Biological Properties of HIV-1 V3 Evolutionary Variants

HIV-1 V3 进化变异体的生物学特性

• 批准号: 9321715
• 负责人: Ronald I Swanstrom
• 金额: $ 39.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321715
• 关键词: Address; Age; Biological; Biological Assay; Biological Process; Biology; Blood; CD4 Positive T Lymphocytes; Cell Count; Cells; China; Clinic; Consensus Sequence; DNA; Data; Data Set; Defective Viruses; Detection; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Disease Management; Disease Progression; Drug resistance; Evolution; Failure; Frequencies; Generations; Genes; Genetic Recombination; Genome; Genomics; Goals; Guidelines; HIV; HIV-1; Hospitals; Human; Human body; Infection; Learning; Length; Link; Mediating; Menopause; Methods; Minor; Monitor; Mutation; Natural History; Nature; Neurons; Patients; Pattern; Plasma; Population; Population Analysis; Population Dynamics; Population Heterogeneity; Problem Solving; Property; Residual state; Resistance; Role; Sampling; Source; Structure; Technology; Testing; Time; Treatment Failure; Variant; Viral; Viral Genes; Viral Genome; Viremia; Virion; Virus; Woman; abstracting; base; biological systems; cell type; clinical care; cohort; deep sequencing; inflammatory marker; insight; neutralizing antibody; new technology; next generation sequencing; pathogen; quantum; resistance mutation; screening; sequencing platform; tool; viral RNA; whole genome

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.

项目摘要/摘要 病毒基因序列代表了有关生物过程的丰富而宝贵的信息来源。 过去十年中，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测病毒种群的多样性。我们最近开发了底漆ID策略 克服了常规NG的几个严重局限 由前面的PCR步骤引入的重组，减少了测序平台的错误，并 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 放。在此应用程序中，我们描述了如何将NGS的底漆ID与NGS一起使用，以解决长期存在的问题 HIV-1种群分析在没有治疗，治疗失败和 进行抑制疗法。此外，我们将将我们的序列数据与最先进的进化分析联系起来 目的是使高质量的数据集和工具易于用于进一步的次要分析。在AIM 1中， 我们将获得28位HIV感染妇女（来自WIHS队列）的纵向等离子体样本。 CD4+ T细胞计数直到它们发展为小于100个细胞/µL的CD4+ T细胞计数。我们将表演 多路复用引物ID测序以获得接近全长HIV-1基因组。我们预测病毒中的X4变体 种群首先以低抽象出现，我们将能够比以前观察到的要早得多。 此外，我们将研究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记链接到创新，中枢神经系统损失以及广度和效能的标记 中和抗体。在AIM 2中，我们将应用多路复用引物ID测序方法 耐药性测试的筛查工具。我们假设耐药性突变的潜力 可以从次要变体中发生中介逃生，太小了，无法可靠地检测到的方法 我们将分析来自＃8的HIV诊所的大量受试者的样本 中国广州的医院，以确定通过评估治疗失败遗漏的频率频率 使用Sanger测序，确定传播或预先存在的突变在WHO的失败中的作用 线路疗法，并在WHO二线治疗后随后的失败后定义突变模式。在AIM 3中， 我们将使用带有底漆ID的深层测序来监测治疗受试者的人群变化。我们 假设低水平病毒种群的变化即使在治疗时也会发生，并且可以是 使用深处监测。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，以定义病毒种群不断变化的其他生物学相关性 随着女性疾病的进展，以定义如何建立病毒群体 次要序列变体的稳态水平，以检查耐药性变异在治疗中的作用 失败，并探索NGS技术在治疗受试者的设置中的实用性。