Aurintricarboxylic Acid Is a Potent Inhibitor of Influenza A and B Virus Neuraminidases

Hashem, Anwar M.; Flaman, Anathea S.; Farnsworth, Aaron; Brown, Earl G.; Van Domselaar, Gary; He, Runtao; Li, Xuguang

Confluent cells in 6 well plates were washed twice with PBS and inoculated with 1 ml MEM containing PR8 for 2 h at 37 uC. Inoculums were aspirated, cells were washed twice with PBS and treated with 1% DMSO, 100 mg/ml ATA, 100 mg/ml AH or 100 mg/ml NAA for 48 h at 37uC. Cells were scraped off wells and collected by centrifugation at 16006g for 5 min. Supernatants were transferred to a new tube, then cells were lysed by 2 cycles of freezing and thawing. Supernatants and lysates were subjected to plaque assay as described previously [26] to determine the extracellular and cell-associated virus titer yield upon ATA treatment. Confluent cell monolayers in 6 well plates were incubated with 1 ml of supernatant or lysate at 37uC for 2 h. The inoculum was removed, cells were washed with PBS and overlaid with the maintenance MEM medium containing 0.8% agarose, 0.2% BSA, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate (pH 7.2), 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mg/ml TPCK-treated trypsin. After incubation for 3 days at 37uC in a humidified atmosphere of 5% CO 2 , cells were either stained with 0.01% neutral red directly or fixed with 10% formaldehyde, followed by staining with 0.5% crystal violet for plaque counting.