of H2DCFDA. Interestingly, after isorhamnetin treatment there was a significant reduction in virus-induced ROS generation. Moreover, isorhamnetin treatment significantly suppressed virus-induced ERK phosphorylation. In vivo antiviral activity of isorhamnetin against the influenza A virus Mice were infected with influenza A/PR/8/34 and isorhamnetin was administered via the intranasal route at a dose of 1 mg/kg/day for 5 days. In addition, we used a control group of mice that were administrated Tamiflu orally at a dose of 10 mg/kg once per day for 5 days. Although previous reports showed no harmful effect of DMSO in mice at less than 1%, we checked any toxic or side effect of 0.2% DMSO in mice and confirmed no significant toxic effect form administration of isorhamnetin or 0.2% DMSO in mice.We compared the effect of isorhamnetin with the effect of Tamiflu against the influenza virus. The 10 / 21 Antiviral Effect of Isorhamnetin against Influenza Antiviral Effect of Isorhamnetin against Influenza 12 / 21 Antiviral Effect of Isorhamnetin against Influenza Fig 3. Measurement of antiviral activity of isorhamnetin by virus yield and HI assays. Viral yield reduction assay was carried out by seeding MDCK cells in a 6-well plate infected with influenza A/PR/8/34 viruses for 2 hr, followed by virus removal, and flavonoids treatment PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763871 in a dose dependent manner for 48 hr. After incubation, a virus yield reduction assay was carried out using media soup. The HA titers were interpreted as HAU/50L. P < 0.05, P <0.01. Hemagglutination inhibition assay: The flavonoids were serially diluted using PBS and were added to an equal volume of the virus. For checking RBCs hemolysis inhibition potency, 50L of 1% chicken RBCs were added to each well of a 96-well plate incubated for 30min at room temperature. P < 0.05, P <0.01. doi:10.1371/journal.pone.0121610.g003 virus titers in lung tissue were measured to evaluate the inhibition of influenza virus replication. We sacrificed 3 mice per group 7 days after infection for the determination of lung viral titers, which were reported as EID50/mL per lung. The virus titers in the lungs of the groups of mice treated with isorhamnetin and Tamiflu were 103.5 and 104.5 EID50/mL, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 respectively, and these values were lower than that of the control group. Moreover, isorhamnetin treatment decreased viral titer after injection in ovo in the embryonated egg. The virus titers for the isorhamnetin and Tamiflu treatments were 108.0 and 108.5 EID50/mL, respectively, and these were lower than that of the control group . These data demonstrated that isorhamnetin decreased viral titer more effectively than Tamiflu. The mice were also monitored daily for 14 days to assess body weight changes and survival rates. Mice treated with isorhamnetin showed reduced body weight loss in comparison with the PBS control group. For the survival rate, although the PBS control group of mice died by day 6after virus infection, the 118414-82-7 web isorhamnetin-treated groups showed a greater survival rate, which ranged from 70 80%. Taken together, these data confirm the in vivo antiviral 
potency of isorhamnetin against influenza A virus infection. Discussion Autophagy is an essential catabolic pathway that is important in the elimination of harmful and unneeded proteins in eukaryotic cells. The link between autophagy and pathogenic infections, including influenza virus infection, has been reported in previous studies. Interestingly, autophagy is involved in the replication