Inhibition of avian-origin influenza A(H7N9) virus by the novel cap-dependent endonuclease inhibitor baloxavir marboxil

Figure 1. Therapeutic effects of BXM on survival and weight loss in a lethal infection model of mice infected with a low dose of the A(H7N9) virus. Mice were intranasally inoculated with 4.0 × 105 TCID50/mouse (10.4 MLD50) of A/Anhui/1/2013 (H7N9) viruses, and treatment was started immediately after virus inoculation (n = 10/group). Survival time (a) and body weight loss (b) were monitored through a 28-day period after the infection. bid (bis in die): twice a day. The log-rank test was applied for comparison of the survival time between each group (*p < 0.05, **p < 0.001 compared to vehicle, †p < 0.01 compared to OSP at 5 mg/kg twice a day, §p < 0.05 compared to OSP at 50 mg/kg twice a day). Dunnett’s multiple-comparison method was applied for statistical analysis of body weight changes (*p < 0.01, **p < 0.001 compared to vehicle, †p < 0.001 compared to OSP at 5 mg/kg twice a day, §p < 0.001 compared to OSP at 50 mg/kg twice a day). The body weights at 5 dpi were calculated from nine mice in vehicle-treated group because one of the ten mice showed more than 30% reduction and was euthanized.

Figure 2. Inhibitory effect of BXM on virus titers in the lungs of mice infected with a low dose of the A(H7N9) virus. Mice were inoculated with 4.0 × 105 TCID50/mouse (10.4 MLD50) of A/Anhui/1/2013 (H7N9) virus and treatment was started immediately after virus inoculation (n = 5/group). The virus titers (TCID50) in lungs of mice at 1, 3 and 5 dpi were measured in MDCK cells. The lower limit of quantification of the virus titer is indicated by a dotted line (1.5 Log10 TCID50/mL). Dunnett’s multiple-comparison method was conducted for statistical comparison (*p < 0.05, **p < 0.001 compared to vehicle, †p < 0.05, ††p < 0.01, †††p < 0.001 compared to OSP at 5 mg/kg twice a day, §p < 0.01, §§p < 0.001 compared to OSP at 50 mg/kg twice a day).

Figure 3. Suppressive effects of BXM on proinflammatory cytokine and chemokine production in the lungs of mice infected with a low dose of the A(H7N9) virus. Mice were intranasally infected with 4.0 × 105 TCID50/mouse (10.4 MLD50) of A/Anhui/1/2013 (H7N9) virus, and treatment was started immediately after virus inoculation (n = 5/group). The proinflammatory cytokines and chemokines, IL-6, MCP-1, MIP-1α and IFN-γ, in the lungs at 1, 3 and 5 dpi were quantified. Dunnett’s multiple-comparison method was employed for the comparison (*p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle).

Figure 4. Effects of BXM on survival, weight loss and virus titers in the lungs in mice infected with a high dose of the A(H7N9) virus. Mice intranasally infected with 1.2 × 106 TCID50/mouse (31.1 MLD50) of A/Anhui/1/2013 (H7N9) virus were administrated with treatment started immediately after virus inoculation, and survival time (a) and body weight loss (b) were analyzed. The log-rank test was applied for survival time (*p < 0.001 compared to vehicle, †p < 0.001 compared to OSP at 5 mg/kg twice a day, §p < 0.001 compared to OSP at 50 mg/kg twice a day). Dunnett’s multiple-comparison method was employed for body weight changes (*p < 0.001 compared to vehicle, †p < 0.001 compared to OSP at 5 mg/kg twice a day, §p < 0.001 compared to OSP at 50 mg/kg twice a day). Body weights at 4 and 5 dpi were calculated from nine mice because one of the ten mice in vehicle-treated group showed more than 30% reduction and was euthanized. (c) The virus titers (TCID50) in the lungs of mice at 1 and 5 dpi were measured. The lower limit of quantification of the virus titer is indicated by a dotted line (1.5 Log10 TCID50/mL). Dunnett’s multiple-comparison method was applied for the comparison (*p < 0.05, **p <…

Figure 5. Effects of delayed treatment of BXM on a low dose of A(H7N9) infection. Mice were intranasally inoculated with 4.0 × 105 TCID50/mouse (10.4 MLD50) of A/Anhui/1/2013 (H7N9) viruses, and BXM treatment was started at (a) immediately, (b) 24 or (c) 48 hours after virus inoculation (n = 5/group). Vehicle or OSP treatment was started immediately after virus inoculation (n = 5/group). Survival time and body weight loss were monitored through a 21-day period after the infection. The shaded area represents the treatment period. The log-rank test was applied for comparison of the survival time between each group (*p < 0.01 compared to vehicle, †p < 0.01 compared to OSP at 5 mg/kg twice a day).