Figure Legends
Figure 1 Dose-dependent effects of aprotinin against influenza viruses in vitro . Madine-Darby canine kidney (MDCK) cells were infected with 100 or 1000 TCID50/ml of (A) A/PR/8/34 (H1N1), (B) A/CA/04/09 (H1N1), (C) A/PH/2/82 (H3N2), (D) A/AB/Kor/CN5/09 (H6N5), (E) A/Ck/Kor/01310/01 (H9N2), (F) A/Bris/10/07 (H3N2), or (G) B/Seoul/32/11 (Yamagata-like lineage) and were treated with varying concentrations of aprotinin (10‒200 nM; n = 3 per dose) for 72 h. Cell viability was measured using the EZ-Cytox reagent, and percent virus inhibition was calculated relative to the uninfected MDCK cell viability (cell-only control). TCID50: median tissue culture infectious dose.
Figure 2 Antiviral effect of aprotinin compared with oseltamivir and cytotoxicity assay. (A) Madine-Darby canine kidney (MDCK) cells were infected with A/PR/8/34 (1000 TCID50/ml) and treated with 60 nM aprotinin or 100 μM oseltamivir. Untreated and uninfected MDCK cells (cell) and untreated infected cells (PR8) were used as controls. (B) To determine the cytotoxicity of aprotinin, the cell viability was measured by treating the MDCK cells with the compound for 72 h and compared with that of untreated control cells. Cell viability was measured using EZ-Cytox. The experiments were performed in triplicate. * P < 0.05, ** P < 0.01, and *** P < 0.001, statistically significant difference between the PR8 and PR8 + oseltamivir or PR8 + aprotinin groups. TCID50: median tissue culture infectious dose.
Figure 3 Aprotinin inhibited the replication of various strains of human influenza A virus in MDCK cells. The replication kinetics of (A) A/PR/8/34 (H1N1), (B) A/CA/04/09 (H1N1), and (C) A/PH/2/82 (H3N2) virus were investigated in Madine-Darby canine kidney (MDCK) cells after treatment with aprotinin and oseltamivir. MDCK cells were infected with influenza virus at an MOI of 0.01 in the presence of aprotinin (60 nM) or oseltamivir (100 μM), and supernatants were harvested at 0, 16, 24, 32, 48, and 64 h. The virus titer in the supernatants was determined by TCID50. *P < 0.05, **P< 0.01, and ***P < 0.001, statistically significant difference between the virus-only (media) group and the aprotinin treatment group. MOI: multiplicity of infection; TCID50: half-maximal tissue culture infectious dose.
Figure 4 Aprotinin inhibited the replication of various strains of avian influenza A virus in MDCK cells. The replication kinetics of (A) A/AB/Kor/CN2/09 (H5N2), (B) A/AB/Kor/CN5/09 (H6N5), and (C) A/Ck/Kor/01310/01 (H9N2) were investigated in Madine-Darby canine kidney (MDCK) cells after treatment with aprotinin and oseltamivir. MDCK cells were infected with influenza virus at an MOI of 0.01 (H5N2 and H9N2) or 0.001 (H6N5) in the presence of aprotinin (60 nM) or oseltamivir (100 μM), and supernatants were harvested at 0, 16, 24, 32, 48, and 64 h. The virus titer in the supernatants was determined by TCID50. *P < 0.05, **P< 0.01, and ***P < 0.001, statistically significant difference between the virus-only (media) group and the aprotinin treatment group. MOI: multiplicity of infection; TCID50: half-maximal tissue culture infectious dose.
Figure 5 Aprotinin inhibited the replication of oseltamivir-resistant influenza viruses in MDCK cells. The replication kinetics of (A) A/Bris/10/07 (H3N2) and (B) B/Seoul/32/11 were investigated in Madine-Darby canine kidney (MDCK) cells after treatment with aprotinin and oseltamivir. MDCK cells were infected with influenza virus at an MOI of 0.01 (H3N2) or 0.001 (B/Seoul/32/11) in the presence of aprotinin (60 nM) or oseltamivir (100 μM), and supernatants were harvested at 0, 16, 24, 32, 48, and 64 h. The virus titer in the supernatants was determined by TCID50. *P< 0.05, **P < 0.01, and ***P< 0.001, statistically significant difference between the virus-only (media) group and the aprotinin treatment group. MOI: multiplicity of infection; TCID50: half-maximal tissue culture infectious dose.
Figure 6 Concentration-dependent reduction in the replication of various influenza virus strains in MDCK cells following treatment with aprotinin. To calculate the half-maximal effective concentrations (EC50) of aprotinin against the different influenza virus strains, Madine-Darby canine kidney (MDCK) cells were infected with (A) A/PR/8/34 (H1N1, 1000 TCID50/ml), (B) A/CA/04/09 (H1N1, 1000 TCID50/ml), (C) A/PH/2/82 (H3N2, 1000 TCID50/ml), (D) A/AB/Kor/CN5/09 (H6N5, 100 TCID50/ml), (E) A/Ck/Kor/01310/01 (H9N2, 1000 TCID50/ml), (F) A/Bris/10/07 (H3N2, 1000 TCID50/ml), and (G) B/Seoul/32/11 (100 TCID50/ml) treated with varying doses of aprotinin (10-200 nM). Three days after virus infection, cell viability was measured using EZ-Cytox, and each data point represents the average of the experiment performed in triplicate cell cultures. TCID50: half-maximal tissue culture infectious dose.
Figure 7 Antiviral effect of aprotinin against influenza A virus in mice. Groups of mice (n = 8 per group) were intranasally infected with A/PR/8/34 (H1N1) virus at three times the 50% mouse lethal dose (3 LD50). Oseltamivir was orally administered twice a day at 10 mg/kg/day, and aprotinin was intravenously administered at 2 mg/kg/day, twice a day for 5 days. (A) Change in body weight of mice and (B) survival rates were monitored for 2 weeks. ***P < 0.001, a statistically significant difference between the negative control group (virus-infected only) and the aprotinin treatment group.