Evaporation-Driven Fluid Flow Enhances Sensitivity of An In Vitro Influenza Virus Drug-Susceptibility 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.  Although 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 traditional “plaque reduction assay” can be used to quantify the ability of drug to inhibit virus spread in infected cell culture monolayers under agar, which inhibits convection flows.  Because there are no flows in this assay, the virus spreads only to neighboring cells.  These circular plaques are counted, and the effectiveness of the drug is quantified as the ability to inhibit plaque formation.  Modifying this assay by culturing infections under liquid media rather than agar gel so that spontaneous natural convection 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 and quantifying comet area rather than simple counts, we have reduced assay time and increased the drug-sensitivity by an order of magnitude relative to the plaque reduction assay. 

We have also shown that the morphology of the comets depends strongly on the relative humidity level during incubation, providing evidence that the driving force behind the spontaneous flows arising in culture wells arise from evaporative cooling at the air-liquid interface.  By confocal imaging of a temperature-sensitive dye solution, we characterized the three dimensional temperature profile within the fluid and predicted the resulting flow patterns using computational fluid dynamics.  The improved sensitivity of flow-enhanced assays allows drug sensitivity assays or screens to be performed with lower drug concentrations, minimizing the toxicity to cells.  In addition, other groups conducting in vitro biological assays should be aware of flow patterns in wells and the affects of small changes in relative humidity.