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

项目摘要 RNA病毒的高突变率和短复制时间导致了快速进化，创造了多样化的 一团紧密相关的遗传变异一种这样的病毒是分段的负链RNA甲型流感病毒 IAV病毒是每年造成全球29万至65万人死亡的流感流行病的罪魁祸首。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群体不能表达来自其八个必需基因片段中至少一个的蛋白质。 传统的批量测定，如噬斑测定，可能无法定量这些非感染性颗粒，这些颗粒是 在病毒动力学、动力学和传播中非常重要。因此，我们的长远目标是发展 一种创新方法，用于更高分辨率地检查病毒的异质性和随机性 单细胞水平的多样性影响种群水平的动态。我们将开发一种新的方法， 液滴qRT-PCR，这将允许精确测量甲型流感病毒（IAV）的病毒产生 从数千个单个感染细胞中分离出来计划中的研究非常适合于理解 IAV在单细胞水平上的种群动态。本项目的具体目标包括：（1）量化 IAV病毒生产或爆发大小分布，从数千个单细胞使用微流体液滴qRT- PCR法(2)开发液滴qRT-PCR数据分析管道，以量化来自 数以千计的随机取样的液滴进行PCR。(3)多重Taq-Man分析技术的发展 使用液滴qRT-PCR在单细胞水平上定量不同IAV毒株的WT和DI流行率。这 这种方法将使我们能够使用低容量和快速的高分辨率来研究IAV的异质性。 吞吐量，从而大大降低成本和速度。我们提议的调查不仅会突破 但也将有助于解剖异质性对病毒疾病的作用， 动态，了解病毒感染的传播和持续性，免疫反应的激活，以及 设计用于疫苗的减毒病毒。

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.