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

Total RNA was extracted from cells 48 h following infection with influenza PR8 and treatment with ATA using an RNeasy mini kit (Qiagen Inc. Valencia, CA). Extracted RNA was treated with DNAse I (Ambion Inc., Streetsville, Ontario, Canada) and single step reverse transcription-PCR was performed using a Titan One Tube RT-PCR kit (Roche Applied Science, 68298 Mannheim, Germany) according to manufacturer's instructions. Influenza PR8 nucleoprotein RNA was amplified from 200 ng RNA in a total reaction volume of 50 ml using the forward and reverse primers 59-ACTCACATGATGATCTGG-39 and 59-CTGCATTGTCTCCGAAGA-39, respectively. Reverse transcription was performed at 50uC for 30 min. After an initial denaturation at 94uC for 2 min, two amplification steps were performed. The first step consisted of 10 cycles of denaturation at 94uC for 30 sec, annealing at 58uC for 30 sec and elongation at 68uC for 45 sec. The second step consisted of 18 cycles under the same cycling conditions, with 5 sec added to the elongation step in each cycle. After a final extension at 68uC for 7 min, 20 ml of the RT-PCR product was loaded on an 1% agarose gel and visualized by SYBR Green staining and UV exposure.