| Viruses cause deadly diseases such as influenza, cancer, hepatitis and acquired immunodeficiency syndrome (AIDS), creating a significant threat to human health. To cause diseases, viruses must reproduce within host cells, and upon release from infected cells, their progeny must spread to and infect other cells. New methods to quantify how viruses grow and how their infections spread may advance our fundamental understanding of virus biology and provide tools for clinical diagnostics and drug testing.;Established methods quantify the production of virus particles by a population of infected cells during a single infection cycle, providing an average yield that masks potentially informative cell-to-cell differences. We studied virus production at the single-cell level by quantifying yields of vesicular stomatitis virus (VSV) from infected baby hamster kidney (BHK-21) cells. Single-cell yields spanned from 8000 to below the detection limit of 10 virus particles. Although viral genetic variation contributed little to the diversity, based on analysis of the amplified virus populations, cells infected at different phases of their growth cycle produced from 1400 (early S phase) to 8700 (G 2M transition) virus particles, accounting for the middle-to-high range of the yield distribution. The remaining low-yield infections may arise from other factors such as stochastic gene expression, a mechanism that has yet to be widely observed for viruses.;The established plaque assay defines conditions for single virus particles to initiate spatially spreading infections over multiple infection cycles in a monolayer of host-cells, producing macroscopic regions of cell death called "plaques". We employed fluid flows to enhance virus spread, producing elongated regions of cell death shaped like comets. Inhibition of comet formation by anti-viral drug (5-fluorouracil), combined with quantitative imaging, provided a measure of drug susceptibility that was nearly 20-fold more sensitive than the established assay. To better control the culture and flow conditions, we implemented this assay in microscale channels, employing passive pumping to drive flows across infected cells. The greater sensitivity, reduction in scale, simplified fluid handling, and image-based quantification make this flow-enhanced infection platform attractive for applications in high-throughput drug screening, sero-conversion testing, and patient-specific diagnosis of viral infections. |
