(13a) Leveraging Fluid Flow for An Enhanced-Sensitivity Virus Infection Assay

Influenza A in the United States causes around 36,000 annual deaths and $10 billion in economic losses. Rapid evolution through multiple mechanisms leads to periodic novel strains with elevated virulence, transmissibility, and/or drug resistance. Though vaccination is the primary public health measure designed to fight influenza, antiviral drugs are very important and effective for individual cases of elevated risk or compromised immune system. The drug susceptibility of virus populations must be very carefully monitored to ensure that patients are proscribed medications effective against their infections. A commonly used in vitro method called the ?plaque reduction assay? quantifies the affect of drug on monolayer cell cultures inoculated with a countable number of infectious virus particles. These cells are then incubated under a layer of semi-solid agar gel, designed to keep the infections localized and spatially separated so the number of resulting ?plaques? ? circular regions of cell death ? can be quantified. The number of plaques is expected to decrease with greater drug concentration. We have modified this assay by culturing in liquid media rather than agar gel so that spontaneous flows arising within the fluid spread the infections in comet-like patterns radiating outward from the center of the plate. By leveraging these spontaneous flows, we have increased the drug sensitivity of the assay by an order of magnitude. Our studies suggest that evaporation-induced thermal gradients may provide the driving force for these radial flow patterns.