Figure 1.
Survival, body weight, AST, ALT, and virus titer in blood and organs of IFNAR−/− mice infected with different doses of CCHFV.
Mice were inoculated i.p. with 10, 100, 1,000, or 10,000 FFU of virus. Organ titers were determined in animals that succumbed to the infection or had to be euthanized due to the severity of the disease. Mean and standard deviation are shown for weight and log-transformed organ titers. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The data for the 100 and 1,000 FFU groups of naïve animals were pooled with corresponding data from placebo controls shown in Fig. 5 to provide more reliable estimates for the parameters. The range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice [59] are shaded in grey. Notes. 10 FFU group: The 2 surviving animals showed neither AST/ALT elevation nor viremia at day 4 (the ALT value for one animal was not determined). 100 FFU group: The animal, which died at day 6, showed no AST/ALT elevation and viremia at days 2 and 4.
Figure 2.
Histopathology and distribution of viral antigen in liver and spleen of naïve and CCHFV-infected IFNAR−/− mice and effect of treatment with ribavirin and T-705.
Animals were inoculated i.p. with 100 FFU of CCHFV. Top: Liver was collected (i) from naïve animals (ii) from animals that succumbed to the infection without treatment at day 3 p.i., (iii) that succumbed to the infection at day 9 p.i. following ribavirin treatment with 100 mg/(kg×d), and (iv) that were euthanized at day 3 p.i. during T-705 treatment with 300 mg/(kg×d). Treatment with ribavirin or T-705 was commenced 1 h p.i. and continued until death or day 8. Bottom: Spleen was collected from naïve animals and animals that succumbed to the infection without treatment at day 3 p.i. Sections were stained with H&E and the distribution of CCHFV in the tissue was visualized using a monoclonal antibody against CCHFV NP. Histopathological findings are representative for 2–3 animals that were analyzed per group. Scale bar = 50 µm.
Figure 3.
IHC for markers of inflammation, apoptosis, and proliferation in the liver of naïve and CCHFV-infected IFNAR−/− mice and effect of treatment with ribavirin and T-705.
Animals were inoculated i.p. with 100 FFU of CCHFV. Liver was collected (i) from naïve animals (ii) from animals that succumbed to the infection without treatment at day 3 p.i., (iii) that succumbed to the infection at day 9 p.i. following ribavirin treatment with 100 mg/(kg×d), and (iv) that were euthanized at day 3 p.i. during T-705 treatment with 300 mg/(kg×d). Treatment with ribavirin or T-705 was commenced 1 h p.i. and continued until death or day 8. Sections were stained with antibodies against CD3, B220, Ly6G, Iba-1, iNOS, cleaved caspase-3, and Ki67. T cells are marked with arrows, as the antibody against CD3 shows some spurious crossreactivity. Histopathological findings are representative for 2–3 animals that were analyzed per group. Scale bar = 50 µm.
Figure 4.
Testing of the antiviral activity of ribavirin, arbidol hydrochloride, and T-705 in cell culture.
Vero E6 cells were inoculated with CCHFV at a MOI of 0.01 and compound was added 1–4 days p.i. by immunofocus assay. Cell growth and viability under compound treatment was determined by the MTT method. Dose–response curves were fitted to the data. The graphs show representative experiments with data points representing mean and standard deviation of ≥3 replicates. The IC50, IC90, and IC99 values were calculated from the sigmoidal functions of 2–3 independent experiments and are shown below the graphs (mean and range).
Figure 5.
Treatment of CCHFV-infected IFNAR−/− mice with ribavirin and arbidol hydrochloride.
Mice were inoculated i.p. with 10, 100, or 1,000 FFU of virus. Ribavirin was administered i.p. once daily. Animals received a ribavirin dose of 100/(kg×d) or 0.9% NaCl as a placebo. Treatment was commenced 1 h p.i. and continued until death or day 8. Arbidol was administered once daily per os using a stomach probe. Animals received an arbidol dose of 150 mg/(kg×d) or 0.5% methylcellulose as a placebo. Treatment was commenced 1 day before infection and continued until death or day 8. Organ titers were determined in animals that succumbed to the infection or had to be euthanized due to the severity of the disease. In addition, three animals from the ribavirin-treated group were randomly euthanized at day 3 p.i. to determine the virus titer in organs (weight, AST, ALT, and viremia data obtained from these mice until day 3 were included in the respective graphs). Mean and standard deviation are shown for weight and log-transformed organ titers. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The duration of treatment in the survival plots, the range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice [59] are shaded in grey. Notes. 100 FFU placebo group: The animal, which died at day 6, showed no AST/ALT elevation and viremia at day 4. 100 FFU ribavirin group: The surviving animal did not show detectable viremia, but AST elevation. Viremia was not determined at day 11. 10 FFU arbidol group: The surviving animal had no AST elevation and viremia at day 4. Some values for days 4, 8, and, 11 were not determined due to insufficient amount of blood.
Figure 6.
Treatment of CCHFV-infected IFNAR−/− mice with T-705 and time-of-addition experiments.
Mice were inoculated i.p. with 100 FFU of virus. T-705 was administered twice daily per os using a stomach probe. Animals received a T-705 dose of 300 mg/(kg×d) or 0.5% methylcellulose as a placebo. Treatment was commenced 1 h, 1 day, or 2 days p.i. and continued until death or day 8. Two animals per treatment regimen were euthanized at day 3 p.i. to determine the virus titer in organs. Mean and standard deviation are shown for weight and temperature. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The duration of treatment in the survival plots, the range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice [59] are shaded in grey.
Figure 7.
Definition of the lowest effective dose of T-705.
Mice were inoculated i.p. with 100 FFU of virus. T-705 was administered twice daily per os using a stomach probe. Animals received a T-705 dose of 30, 15, or 7.5 mg/(kg×d) or 0.5% methylcellulose as a placebo. Treatment was commenced 1 h p.i. and continued until death or day 8. The virus titer in organs was determined for animals that succumbed to the infection and for 3 animals of the 30 mg/(kg×d) group that were euthanized at day 3. Organ analysis was not done for the 15 mg/(kg×d) group (n.d.). Mean and standard deviation (or range for n = 2) are shown for weight, temperature and log-transformed organ titers. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The duration of treatment in the survival plots, the range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice [59] are shaded in grey. Notes. Placebo group: The animal, which died at day 5, showed no AST/ALT elevation and viremia at day 3. 7.5 mg/(kg×d) T-705 group: The surviving animal showed neither AST/ALT elevation nor viremia at any time.
Figure 8.
Testing of the antiviral activity of combination of ribavirin and T-705 in cell culture.
Vero E6 cells were inoculated with CCHFV at a MOI of 0.01 and the compounds were added 1×8 drug combination matrix was designed as described in Materials and Methods. Concentration in cell culture supernatant of infectious virus particles was measured 3 days p.i. by immunofocus assay. Cell growth and viability under compound treatment was determined by the MTT method. (A) Dose–response surface for combination of ribavirin and T-705. Sigmoidal dose–response curves were fitted to the single-drug data and the IC50, and IC90 values for ribavirin and T-705 were calculated from the functions. (B) In analogy to the MacSynergy II program [44], [45], a three-dimensional approach was used to identify areas where observed effects are greater (synergy) or less (antagonism) than predicted by the Bliss independence model. To this end, the ratio between predicted virus titer and observed virus titer was calculated for each drug combination. A ratio >1 indicates synergy (i.e. the virus titer predicted for additive effect is higher than the experimentally determined virus titer), a ratio <1 indicates antagonism (i.e. for the virus titer for predicted additive effect is lower than the experimentally determined virus titer). Drug combinations showing a clear difference between experimental and predicted titer (≥4-fold) are marked in yellow with the ratio indicated in red. MTT values are shown in blue for each drug combination.
Figure 9.
Treatment of mice with a combination of ribavirin and T-705.
Mice were inoculated i.p. with 100 FFU of virus. T-705 was administered twice daily per os using a stomach probe. Ribavirin was administered i.p. once daily. Animals received a T-705 dose of 30 or 7.5 mg/(kg×d) in combination with a ribavirin dose of 100 mg/(kg×d). The placebo controls are shown in Fig. 7, as both experiments have been conducted in parallel. Treatment was commenced 1 h p.i. and continued until death or day 8. Mean and standard deviation are shown for weight and temperature. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The duration of treatment in the survival plots, the range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice [59] are shaded in grey. Notes. The two animals that died at day 6 had no detectable virus in organs or blood and no AST/ALT elevation.