Development of Single-Cell Techniques to Investigate Heterogeneity in Virus-Cell Interactions

Viruses cause many human diseases including the flu (influenza A), liver cancer (hepatitis C), and AIDS (HIV). A significant obstacle in developing treatments for viruses---particularly RNA viruses---is that they mutate rapidly and are able to escape the inhibitory effects of anti-viral drugs or vaccines. The resulting genetic diversity does not only allow viruses to escape treatments at the whole organism level, but is also relevant at the cellular level. Viruses have evolved a multitude of mechanisms to evade certain aspects of the innate immune response, which has in turn developed into a multifaceted, highly regulated, and redundant system that utilizes several pathogen recognition mechanisms.;The complexity of virus-host interactions is difficult to understand and characterize, particularly because there is so much cell-to-cell variability that cannot be resolved using standard population-based assays. Technological advances, particularly in live-cell microscopy have allowed researchers to make single-cell measurements that capture this variability. Here, we present one such method that utilizes a microwell based cell-culture environment, combined with a dual-color reporter system, and also employs automated imaging and high-throughput image processing and data analysis techniques to study isolated infections. Our system allows for frequent, sensitive, and quantitative measurements that provide a good representation of the state of infection and innate immune activation in isolated cells.;We employed these new methods to investigate the effects of the innate immune response and the effects of DIP co-infections on VSV replication. Using these methods we found that a small, but potentially important sub-population of cells became activated prior to our detection of infection. These fast acting cells may be important for warning their neighbors of infection, and we explored these affects by treating cells with IFN- beta before infection. Combining information gathered from multiple measures on single-cells, we identified various ways ISGs may be targeting VSV replication. Finally, by studying co-infections of single-cells with DIPs and viable VSV, we found that most cells produced infectious virus although one DIP is considered sufficient to eliminate infectious virus production. These results demonstrate that by harnessing their natural heterogeneity, we can gain insight into the complexity of virus-host interactions.