(216a) 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.