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.

项目概要 RNA病毒的高突变率和短复制时间导致快速进化，创造了多样化的病毒 密切相关的遗传变异云。其中一种病毒是分段负链 RNA 甲型流感病毒 病毒 (IAV)，它是每年流感流行的罪魁祸首，导致全球 290,000 至 650,000 人丧生。 IAV 感染涉及异质的、动态的病毒群体与不同的宿主细胞群体相互作用。 值得注意的是，70-99% 的 IAV 群体无法表达其八个必需基因片段中至少一个的蛋白质。 传统的批量检测（例如噬菌斑检测）可能无法量化这些非感染性颗粒，这些颗粒是 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 创新方法，以更高分辨率检查病毒的异质性和随机性 单细胞水平的多样性影响群体水平的动态。我们将开发一种新方法，微流控 液滴 qRT-PCR，将能够精确测量甲型流感病毒 (IAV) 的病毒产量 来自数千个单一感染细胞。计划中的研究特别适合于理解 单细胞水平的 IAV 种群动态。本项目的具体目标包括：（1）量化 使用微流体液滴 qRT-从数千个单细胞中观察 IAV 病毒的产生或爆发大小分布 PCR。 (2) 开发液滴 qRT-PCR 数据分析流程，以量化单细胞突发大小 数千个随机采样的液滴正在进行 PCR。 (3) 多重Taq-man分析的发展 使用液滴 qRT-PCR 在单细胞水平量化不同 IAV 毒株的 WT 和 DI 流行率。这 该方法将使我们能够使用低容量和快速的高分辨率研究 IAV 异质性 吞吐量，从而大大降低成本和速度。我们提出的调查不仅会突破界限 单细胞病毒学，但也有助于剖析异质性对病毒性疾病的作用，以建立感染模型 动态，了解病毒感染的传播和持续性、免疫反应的激活，以及 用于疫苗的减毒病毒的设计。