High-throughput droplet qRT-PCR microfluidic platform for quantification of virus from single cells

用于定量单细胞病毒的高通量液滴 qRT-PCR 微流控平台

• 批准号: 10387693
• 负责人: Connie B Chang
• 金额: $ 14.6万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-05 至 2022-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387693
• 关键词: Address; Affect; Antiviral Agents; Area; Attenuated; Biological Assay; California; Cell Survival; Cells; Complement; Data; Defective Viruses; Detection; Development; Disease model; Dissection; Drops; Environment; Essential Genes; Event; Evolution; Genes; Genomics; Goals; Heterogeneity; Immune; Immune response; In Vitro; Individual; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza A virus; Investigation; Kinetics; Libraries; Measurement; Measures; Methodology; Methods; Microfluidics; Mutate; Mutation; Outcome; Output; Plaque Assay; Play; Population; Population Dynamics; Population Heterogeneity; Prevalence; Production; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA Viruses; Recovery; Research; Resolution; Role; Sampling; Speed; Testing; Time; Vaccines; Variant; Viral; Virus; Virus Diseases; Virus Replication; base; co-infection; cost; curve fitting; data analysis pipeline; design; experimental study; extracellular; fitness; genetic variant; immune activation; influenza epidemic; influenza infection; innovation; insight; kinetic model; man; novel; novel strategies; particle; pathogen; response; screening; single cell sequencing; stressor; tool; transmission process; virology; virus host interaction

PROJECT SUMMARY The high mutation rates and short replication times of RNA viruses leads to rapid evolution, creating a diverse cloud of closely related genetic variants. One such virus is the segmented, negative stranded RNA influenza A virus (IAV), which is responsible for annual flu epidemics that claim 290,000 to 650,000 lives worldwide. IAV infection involves a heterogeneous, dynamic viral population interacting with a diverse population of host cells. Notably, 70-99% of IAV populations fail to express proteins from at least one of its eight essential gene segments. Traditional bulk assays, such as the plaque assay, can fail to quantify these non-infectious particles, which are highly important in viral kinetics, dynamics and transmission. Thus, our long-term objective is to develop innovative methodology towards a higher resolution examination of how heterogeneity and stochasticity of viral diversity at the single cell level affects population-level dynamics. We will develop a novel method, microfluidic droplet qRT-PCR, that will allow enable the precise measurement of viral production of influenza A virus (IAV) from thousands of single infected cells. The planned research is uniquely suited to enable the understanding of IAV population dynamics at the single cell level. The specific aims of this project include: (1) The quantification of IAV viral production, or burst size distribution, from thousands of single cells using microfluidic droplet qRT- PCR. (2) The development of a droplet qRT-PCR data analysis pipeline to quantify single cell burst size from thousands of randomly sampled drops undergoing PCR. (3) The development of multiplexed Taq-man analysis to quantify WT and DI prevalence for different IAV strains at the single cell level using droplet qRT-PCR. This approach will enable us to investigate IAV heterogeneity in high-resolution using low volumes and rapid throughput, thus greatly reducing cost and speed. Our proposed investigations will not only push the boundaries of single cell virology but will also aid in dissecting the role of heterogeneity on viral disease for modeling infection dynamics, understanding the spread and persistence of viral infections, the activation of immune responses, and the design of attenuated viruses for vaccines.

项目摘要 RNA病毒的高突变率和短复制时间会导致快速发展，从而创造了多种多样的 紧密相关的遗传变异云。一种这样的病毒是分割的，负链的RNA流感A 病毒（IAV），该病毒负责年度流行病的年度流行病，在全球范围内拥有290,000至650,000人。 IAV 感染涉及异质的动态病毒种群与宿主细胞种群相互作用。 值得注意的是，70-99％的IAV种群未能从其八个基本基因段中的至少一种表达蛋白质。 传统的散装测定，例如斑块测定，可能无法量化这些非感染粒子 在病毒动力学，动力学和传播中非常重要。因此，我们的长期目标是发展 创新的方法论，以对病毒的异质性和随机性进行更高的分辨率检查 单细胞水平的多样性会影响人群水平的动态。我们将开发一种新颖的方法，微流体 液滴QRT-PCR，这将允许精确测量流感病毒病毒（IAV）的病毒产生 来自成千上万的单一感染细胞。计划的研究非常适合使人们能够理解 单细胞水平的IAV种群动力学。该项目的具体目的包括：（1）量化 使用微流体液滴QRT-从数千细胞中产生的IAV病毒生产或爆发尺寸分布 pcr。 （2）开发液滴QRT-PCR数据分析管道，以量化单细胞突发的大小 经历PCR的数千个随机抽样。 （3）多路复用TAQ-MAN分析的发展 使用液滴QRT-PCR来量化单细胞水平上不同IAV菌株的WT和DI患病率。这 方法将使我们能够使用低体积和快速的高分辨率研究IAV异质性 吞吐量，从而大大降低了成本和速度。我们提议的调查不仅会突破界限 单细胞病毒学，但也将有助于剖析异质性在病毒疾病中的作用 动态，了解病毒感染的传播和持久性，免疫反应的激活以及 疫苗减毒病毒的设计。