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

Neutral red is a vital dye that is incorporated into the vacuoles of viable cells, can be detected spectrophotometrically and is used to determine cell viability. The in vitro efficacy and toxicity of antiviral compounds have previously been determined by measuring the uptake of neutral red [23] [24] [25] , therefore a neutral red assay kit (Sigma, St. Louis, USA) was used to assess the cytotoxicity and antiviral potential of ATA according to manufacturer's instructions. To determine ATA cytotoxicity, confluent MDCK monolayers in 24-well plates were washed twice with PBS then exposed to ATA in 1 ml post-adsorption medium for 48 h at 37uC, 5% CO 2 . To determine inhibitory potential of ATA alone or in combination with AH, inoculums were aspirated following viral adsorption, cells were washed twice with PBS, then incubated in 1 ml post-adsorption medium containing ATA and/or AH for 48 h at 37uC, 5% CO 2 . DMSO concentration was the same in all treatments. After 48 h, neutral red dye was added to the medium in each well at a concentration of 0.033% and incubated for 2 h at 37uC. The medium was then removed, and cells were fixed by rinsing with 0.1% CaCl 2 in 0.5% formaldehyde. The incorporated dye was solubilized in 1 ml of 1% acetic acid in 50% ethanol. Cells were incubated at room temperature for 10 min with gentle agitation and the absorbance was measured at 540 nm with a Synergy TM 2 Multi-Mode Microplate Reader. MDCK cells were also evaluated by microscopic examination for cytopathic effect (CPE). Results for ATA cytotoxicity were presented as percentage of absorbance of ATA-treated cells relative to that from untreated controls. ATA and/or AH protection results were expressed as percentage of absorbance of treated or untreated infected cells relative to that from untreated, uninfected controls. The concentration of drug required to inhibit 50% of the CPE induced by PR8 (EC 50 ) was determined.