Treatment with the nucleoside analog T-1106 was previously shown to be effective in a hamster model of yellow fever virus (YFV) disease, even though it had only slight activity in cell culture. In the study described in this report, the activity of T-705, a chemically related compound currently undergoing clinical trials for the treatment of influenza (FDANews 4:1, 2007), was tested against YFV in cell culture and in the hamster model. The antiviral efficacy of T-705 in cell culture occurred at a concentration of 330 M, which was more than threefold lower than the concentration at which T-1106 had antiviral efficacy, as determined by a virus yield reduction assay and confirmed by a luciferase-based ATP detection assay. Time-of-addition studies revealed that addition of T-705, T-1106, or ribavirin at 0, 4, 8, or 12 h after virus challenge was effective in inhibiting virus in Vero cells, suggesting that these three agents have similar mechanisms of action in cell culture. Because of its more potent activity in cell culture, it was anticipated that T-705 treatment of hamsters infected with YFV would result in protection from disease. Significant improvements in survival and disease parameters were seen in infected animals when T-705 was administered orally at a dose of 200 or 400 mg/kg of body weight per day when it was given twice a day for 8 days. Significant improvements were also observed with a dose of 400 mg/kg/day when treatment initiation was delayed as late as 3 days after virus inoculation. Although the dose of T-705 required for efficacy in hamsters is higher than that of T-1106 required for efficacy, T-705 treatment is effective in significantly improving disease parameters in YFV-infected hamsters, which may indicate its potential utility in the treatment of YFV disease in humans.

The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.

Antiviral compounds were evaluated for efficacy against yellow fever virus (YFV) in a hamster model of YFV-induced liver disease. Challenge with a 10 2 50% cell culture infectious doses of YFV resulted in a 50-80% mortality rate in female hamsters. Virus was detected by QRT-PCR in liver, kidney, spleen, and serum with peak titers on 4-6 days post-viral challenge (dpi). Serum levels of alkaline phosphatase, alanine aminotransferase (ALT), bilirubin, blood urea nitrogen, potassium, and creatinine were significantly elevated, while serum levels of albumin, amylase, glucose, calcium, globulin, phosphorus, sodium, and total protein were significantly reduced. Packed cell volume and white blood cell count were significantly elevated during the course of the infection. Intraperitoneal treatment of hamsters with 0.5-5 μg/kg/d interferon (IFN) alfacon-1, 100 mg/kg/d viramidine, or 50 mg/kg/d ribavirin, initiated 4 h prior to YFV challenge, resulted in significant improvement in survival and serum ALT levels. Treatment with IFN alfacon-1 or ribavirin starting 2 dpi, also significantly improved survival and serum ALT levels in hamsters challenged with YFV. Pre-and post-virus exposure treatment with IFN alfacon-1 was efficacious in improving disease in YFVinfected hamsters.

Yellow fever virus (YFV) causes 30,000 deaths worldwide, despite the availability of a vaccine. There are no approved antiviral therapies for the treatment of YFV disease in humans, and, therefore, these studies were designed to investigate the anti-YFV properties of T-1106, a substituted pyrazine, in a hamster model of YFV disease. Intraperitoneal (i.p.) treatment with 100 mg/kg of body weight/day of T-1106 starting 4 h prior to virus inoculation and continuing twice daily through 7 days post-virus inoculation (dpi) resulted in significantly improved survival, alanine aminotransferase levels in the serum, weight gain, and mean day to death. Virus titer in the liver at 4 dpi was significantly reduced in treated animals, as determined by both quantitative real-time PCR and infectious cell culture assay. No toxicity (weight loss or mortality) was observed at a dose of 100 mg/kg/day in sham-infected control animals. The observed minimal effective dose of T-1106 was 32 mg/kg/day administered either by oral or i.p. treatment. Therapeutic treatment was effective in significantly improving survival when T-1106 was administered beginning as late as 4 days after virus challenge with twice-daily treatment for 8 days at a dose of 100 mg/kg/day. With favorable safety, bioavailability, and postviral challenge treatment efficacy, T-1106 was effective in the treatment of disease in hamsters infected with YFV and should be further studied for potential use as a therapy for human YFV disease.

Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the D-(-) enantiomer of carbodine [(-)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 μg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the L-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (-)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/kg/d resulted in significant improvement in survival, virus load in the brain, weight change, and mean day to death in a mouse model of TC-83 VEEV disease. A single dose of (-)-Carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (-)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (-)-carbodine is effective when initiated after the establishment of brain infection.