Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment

Abstract

Rift Valley fever is a zoonotic, arthropod-borne disease that affects livestock and humans. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus) is primarily transmitted through mosquito bites, but can also be transmitted by exposure to infectious aerosols. There are presently no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. In addition, efficacy has also been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, hamsters challenged with the highly pathogenic ZH501 strain of RVFV were used to evaluate the activity of favipiravir against lethal infection. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days of infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater of hamsters challenged with RVFV when administered within 1 or 6h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden observed in the brain in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

Keywords: Bunyavirus; Favipiravir (T-705); Phlebovirus; Ribavirin; Rift Valley fever virus.

Figure 1. In vitro activity of T-705 against the ZH501 strain of RVFV

Vero 76 cell cultures were infected with RVFV, treated with various concentrations of T-705 or ribavirin, and A) the inhibition of viral replication was determined by endpoint titration of the culture supernatants. B) Cytotoxicity of the compounds was determined by neutral red dye uptake to measure cell viability in cultures of uninfected cells treated in parallel. Cytotoxicity data represent the percent cell viability after a 3-day incubation compared to untreated controls. The data are representative of 3 independent experiments and reflect the mean and standard deviations from triplicate samples.

Figure 2. T-705 post-RVFV exposure treatment protects hamsters from lethal disease

Hamsters challenged s.c. with 30 PFU of RVFV-ZH501 received the indicated doses of T-705, ribavirin, or placebo (p.o., twice daily) beginning 1 h post-infection. A) Survival outcome and day 3 B) serum, C) liver, and D) spleen virus titers from animals infected and treated in parallel are shown. All animals from the 20 mg/kg/day T-705 and placebo treatment groups, and one hamster from the 60 mg/kg/day T-705 treatment group, succumbed prior to sacrifice. Unique symbols in each treatment group represent values for the same animal in B-D. For percent survival, **P < 0.01 and ***P < 0.001 compared to placebo; _bP < 0.01 compared to animals treated with ribavirin. For viral titers, *P < 0.05 and **P < 0.01 compared to animals treated with 60 mg/kg/day T-705.

Figure 3. Sub-acute central nervous system RVFV infection in ribavirin-treated animals that survive the acute disease

Hamsters were treated as described in Figure 1. Two animals in the ribavirin-treated group were found to be moribund on day 9 post-infection and were sacrificed for analysis of serum, liver, spleen, and brain virus titers, and histopathology. A) Analysis of viral titers in moribund RVFV-infected hamsters treated with ribavirin. Histopathologic findings in the cerebrum display B) multifocal neuronal necrosis and neuronophagia and C) neuropil necrosis. H&E staining, bars = 30 µm.

Figure 4. T-705 intervention is effective out to 6 h post-RVFV exposure

Hamsters were treated with T-705 (200 mg/kg/day), ribavirin (75 mg/kg/day), or placebo, twice-daily p.o. for 14 days beginning 1, 6 or 24 h post-infection (hpi). A) Percent survival and day 2 B) serum, C) liver, and D) spleen virus titers are shown. Five animals from varying placebo groups and one animal from the T-705 24 hpi group succumbed prior to sacrifice. Unique symbols in each treatment group represent values for the same animal in B-D. *P < 0.05 and ***P < 0.001 compared to respective placebo groups; ^bP < 0.01 and ^cP < 0.001 compared to animals treated with ribavirin.

Figure 5. Analysis of brain viral titers in moribund RVFV-infected hamsters treated with T-705 or ribavirin

Hamsters were treated as described in Figure 4. Five animals were discovered moribund and euthanized on the indicated day (d) post-infection for analysis of virus titers and histopathology. A) Brain viral titers in moribund RVFV-infected hamsters treated with T-705 (blue symbol) or ribavirin (red symbols). Histopathologic analysis of cerebrums displayed B) perivascular mixed inflammatory cell infiltration, C) neutrophilic choroiditis and ventriculitis, D) neutrophilic meningitis and E) neuropil vasculitis and hemorrhage. H&E staining, bars = 20 µm.

Figure 6. Combined T-705 and ribavirin therapies significantly improve survival outcome and reduce viral burden when starting treatment 24 h post-RVFV challenge

Hamsters were treated p.o. with T-705, ribavirin, or a combination of both compounds starting 24 h post-infection (see Table 1 for detailed description of the treatment regimens). A) Percent survival and day 2 B) serum, C) liver, and D) spleen virus titers are shown. One animal each in the T-705 monotherapy and placebo-treated group succumbed prior to sacrifice on day 2. Unique symbols in each treatment group represent values for the same animal for B-D. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to placebo-treated animals; ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared to animals receiving T-705 monotherapy; ^xP < 0.05, ^zP < 0.001 compared to hamsters treated only with ribavirin. H (high dose), L (low dose), LD (loading dose).