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

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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.