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.