Tilorone-Dihydrochloride Protects against Rift Valley Fever Virus Infection and Disease in the Mouse Model

Abstract

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East that can affect humans and ruminant livestock. Currently, there are no approved vaccines or therapeutics for the treatment of severe RVF disease in humans. Tilorone-dihydrochloride (Tilorone) is a broad-spectrum antiviral candidate that has previously shown efficacy against a wide range of DNA and RNA viruses, and which is clinically utilized for the treatment of respiratory infections in Russia and other Eastern European countries. Here, we evaluated the antiviral activity of Tilorone against Rift Valley fever virus (RVFV). In vitro, Tilorone inhibited both vaccine (MP-12) and virulent (ZH501) strains of RVFV at low micromolar concentrations. In the mouse model, treatment with Tilorone significantly improved survival outcomes in BALB/c mice challenged with a lethal dose of RVFV ZH501. Treatment with 30 mg/kg/day resulted in 80% survival when administered immediately after infection. In post-exposure prophylaxis, Tilorone resulted in 30% survival at one day after infection when administered at 45 mg/kg/day. These findings demonstrate that Tilorone has potent antiviral efficacy against RVFV infection in vitro and in vivo and supports further development of Tilorone as a potential antiviral therapeutic for treatment of RVFV infection.

Keywords: Bunyavirales; Phenuiviridae; Rift Valley fever virus; Tilorone-dihydrochloride; Viral Hemorrhagic fever; broad-spectrum antiviral.

Figure 1

In vitro dose-response of Tilorone against RVFV. Vero CCL81 cells (A,C) or A549 cells (B,D) were infected with RVFV MP12 (A,B) or RVFV ZH501 (C,D) at an MOI of 0.1 for 1 h. Cell culture media with serial 10-fold dilutions of Tilorone was added at 1 h post infection (HPI). Reduction in virus yield was determined by plaque assay in cell culture supernatant collected at 24 HPI. Data is representative of two individual experiments, each with three biological replicates.

Figure 2

Delayed treatment in vitro efficacy of Tilorone against RVFV infection. A549 cells or Vero CCL81 cells were infected with RVFV MP12 (A) or ZH501 (B) at an MOI of 0.1 and treated with 50 μM of Tilorone at the time points indicated. Virus titer in cell culture supernatant was evaluated by plaque assay at 24 h after infection. The dotted line represents the limit of detection in the plaque assay. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

In vivo antiviral efficacy of Tilorone at different doses against RVFV infection in BALB/c mice. Mice (n = 10/group) were infected with 100 PFU RVFV ZH501 via the intraperitoneal (IP) route. Treatment with either 30 mg/kg/day or 60 mg/kg/day of Tilorone was initiated either 24 h before infection or immediately after infection. Tilorone or vehicle solution was administered once daily via the IP route for 9 days after initiation of treatment. (A) Survival of animals receiving Tilorone or vehicle. (B) Percent weight change of animals receiving Tilorone or vehicle. (C) Terminal viremia for euthanized moribund animals receiving Tilorone or vehicle. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 3

In vivo antiviral efficacy of Tilorone at different doses against RVFV infection in BALB/c mice. Mice (n = 10/group) were infected with 100 PFU RVFV ZH501 via the intraperitoneal (IP) route. Treatment with either 30 mg/kg/day or 60 mg/kg/day of Tilorone was initiated either 24 h before infection or immediately after infection. Tilorone or vehicle solution was administered once daily via the IP route for 9 days after initiation of treatment. (A) Survival of animals receiving Tilorone or vehicle. (B) Percent weight change of animals receiving Tilorone or vehicle. (C) Terminal viremia for euthanized moribund animals receiving Tilorone or vehicle. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4

Therapeutic efficacy of Tilorone against RVFV infection in BALB/c mice. Mice (n = 10/group) were infected with 100 PFU RVFV ZH501 via the intraperitoneal (i.p.) route. Treatment of Tilorone was initiated immediately after infection, 1 DPI, 2 DPI, or 3 DPI. Tilorone at 45 mg/kg/day or vehicle was administered once daily via the IP route for 8 consecutive days after initiation of treatment. (A) Survival of animals receiving Tilorone or vehicle. (B) Percent weight change of animals receiving Tilorone or vehicle. (C) Terminal viremia for euthanized moribund animals receiving Tilorone or vehicle. *** p < 0.001.

Figure 5

Histopathological changes reduced in Tilorone-treated mice. Formalin fixed tissues were embedded in paraffin and processed for H&E staining. Images represent livers of vehicle control and tilorone-treated mice. (A) Ballooned hepatocytes in vehicle-treated control mice at late stage. (B) Hepatocyte necrosis in vehicle-treated control mice. (C) Normal liver from Tilorone-treated survivor. (D) Erythroid cells in liver from Tilorone-treated survivor. (E) Megakaryocyte in liver from Tilorone-treated survivor. (F) Neutrophil infiltrate in liver from Tilorone-treated survivor.