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.
