Quantifying progeny production from individual influenza virus-infected cells

Abstract

The distribution of progeny virions produced by virus-infected cells is extremely heterogeneous. This trend has been observed in diverse viruses, including bacteriaphage and pathogenic human viruses. To date, it has been difficult to explain why some infected cells generate thousands of progeny virions while others – infected under identical conditions – produce no progeny.Established methods for quantifying progeny from single cells rely on the isolation of individual cells during infection. Such methods are not compatible with contemporary single-cell assays. With limited information gathered about the host and virus processes that occur during infection, the factors that might influence progeny production at the single-cell level have remained largely inaccessible. I have developed new methods to quantify the amount of progeny produced by single influenza virus-infected cells; these methods do not require single-cell isolation during in- fection. Applying these methods, I have simultaneously measured viral transcription, viral genotype, and progeny virion production in the same influenza-infected cells. The correlation between viral transcription and progeny production is surprisingly poor at an early time of influenza infection. Using the viral gene expression information provided by single-cell RNA sequencing, I learned that cells with extremely high viral transcription often lack the influenza non-structural (NS) gene, precluding them from contributing infectious progeny. While this system was developed to study influenza virus progeny production in single cells, individually-traceable virions may be useful in several areas of virology. The general approach to generate highly-diverse libraries of barcoded virions could likely be applied to other viruses with established reverse genetics systems. In the course of developing these methods, further opportunities for optimization have become apparent. I have outlined potential future applications that could be facilitated by either the current virus libraries or more diverse libraries that are developed in the future.