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

项目总结 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 一大堆密切相关的基因变异。其中一种病毒是分段的负链RNA甲型流感病毒 病毒(IAV)，每年导致全球29万至65万人死亡的流感疫情。IAV 感染涉及到一个异质的、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基本基因片段中的至少一个基因片段的蛋白质。 传统的大宗化验，如菌斑化验，可能无法量化这些非传染性颗粒，这些颗粒是 在病毒动力学、动力学和传播方面非常重要。因此，我们的长远目标是发展 对病毒的异质性和随机性进行更高分辨率检查的创新方法 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，微流控 滴状qRT-PCR，这将使精确测量甲型流感病毒(IAV)的病毒产量成为可能 从数以千计的单个感染细胞中分离出来。计划中的研究特别适合于使人们能够理解 单细胞水平上的IAV种群动态。本项目的具体目标包括：(1)量化 使用微流控液滴qRT-从数千个单细胞中产生IAV病毒，或突发大小分布- 聚合酶链式反应。(2)一种液滴qRT-PCR数据分析管道的开发，用于从 对数千个随机抽样的液滴进行了聚合酶链式反应。(3)多重Taq-man分析的发展 用Droplet qRT-PCR在单细胞水平上定量检测不同IAV株的WT和DI患病率。这 该方法将使我们能够在高分辨率下使用低体积和快速 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模型感染中的作用 动力学，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。

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.