Tamiflu

Folin-Ciocalteu’s phenol reagent, vanillin, saponin from quillaja bark, and (+)-catechin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan) and sodium carbonate was purchased from Samchun Chemical (Pyeongtaek, South Korea). Tamiflu were obtained from Roche (Seoul, Korea). B. juncea (seed), F. suspensa, (fruit) and I. britannica (seed) were obtained from Kyungdong-Market in Seoul, Korea. B. juncea, F. suspensa, and I. britannica were authenticated by Professor Hyun-Dong Paik at the Laboratory of Biotechnology (Konkuk University, Seoul, Korea) and stored as voucher specimen KU-H13, KU-H22 and KU-H26, respectively.

The NA activity of pH1N1 influenza A virus (A/Hamburg/NY1580/2009) was measured by a fluorescence-based assay using the fluorogenic substrate 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA, Sigma-Aldrich) which is cleaved by the viral NA enzyme to release the fluorescent product 4-methylumbelliferone (4-MU)^{98 (link)}. Therefore, recombinant wildtype (wt) or NA mutant (D199N, D199E, S247N, S247G) pH1N1 viruses were diluted to an equivalent NA activity and pre-incubated with 10, 100 or 1000 nM Oseltamivir (Tamiflu, Roche) or mock treated with 1xPBS for 15 min at 37 °C. The NA enzyme activity was initiated by adding 40 µM 4-MU-NANA in calcium-TBS (6.8 mM CaCl2, 0.85% NaCl, 0.02 M Tris; pH 7.3). After 30 min at 37 °C, the reaction was stopped by the addition of 100 µl of 0,1 M glycine buffer (pH 10,7) containing 25% ethanol. The fluorescence of released 4-MU was determined with a Safire 2 multi-plate reader (Tecan) using excitation and emission wavelengths of 355 nm and 460 nm, respectively. The mean fluorescence signal of the substrate without virus was subtracted as background from the signals obtained in the other wells. The specific NA activity was expressed in percentage.

In two experiments, ferrets were treated with oseltamivir (Tamiflu, Roche). The drug was dissolved in sterile water to 12 mg/ml, and delivered to ferrets by oral gavage twice daily. Doses of 5 mg/kg/day and 10 mg/kg/day are equivalent to human doses of 75 and 150 mg/day, respectively [29] (link), and represent prophylactic and therapeutic doses. Prophylactic dosing commenced 2 hr prior to infection; therapeutic dosing commenced 6 hr post-infection. In both experiments, dosing continued for 5 days.

Different groups of mice were administered with 100 µL of oseltamivir (Tamiflu; Roche Australia, Sydney, Australia) diluted in phosphate buffered saline (PBS) via oral gavage (o.g.) at 150 mg/kg (once daily) or 20 mg/kg (twice daily), etanercept (Enbrel; Pfizer Australia, Sydney, Australia) administered i.p. at 2.5 mg/kg diluted in PBS or S3I-201 (STAT3 inhibitor VI, Sigma-Aldrich Pty Ltd., Macquarie, Australia; cat. no. 573102) administered i.p. at 5 mg/kg after the onset of disease signs. S3I-201 was first dissolved in DMSO at 200 mg/mL and then further diluted in PBS to a working stock solution of 1 mg/mL.