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 inoculated with PR8 virus for 2 h at 37uC, then treated with DMSO, 100 mg/ml ATA, 100 mg/ml AH in post-adsorption medium for 24 h at 37uC. Cells were scraped off wells and centrifuged at 16006g for 5 min. Medium was discarded and cells were incubated with ice-cold fixative (2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4) for 50 min, with gentle agitation. Cells were pelleted by centrifugation at 20,0006g for 2 min at room temperature. Cell pellets were re-suspended in 0.5 ml fixative then rinsed in 0.5 M cacodylate buffer twice for 10 min and post-fixed with 2% osmium tetroxide for 2 h. The fixed cells were washed with water twice for 10 min, dehydrated with increasing concentrations of ethanol from 50 to 100% and embedded in spurr resin. Thin (70-80 nm) sections were cut on an ultramicrotome and counter stained with uranyl acetate and lead citrate. The sections were viewed and photographed on a JEOL 1010 transmission electron microscope.