General procedure for in vivo antiviral experiments.
Mice (13 to 16 g) were anesthetized by intraperitoneal injection of ketamine-rompun solution and exposed to 100 μl of virus by intranasal instillation. The viral dose utilized was an approximately 90% lethal dose, which was equivalent to 102.3 of the 50% cell culture infectious dose in the case of H1N1 virus and 102.7 of the 50% cell culture infectious dose in the case of H6N2 virus. In the prophylaxis model, drug was administered 4 h before viral infection and 4 h after infection, and treatment continued twice a day (BID) for a total of 5 days; in the treatment model, drug was given at times indicated after the viral infection. Each infected group contained 7 to 10 mice, and normal controls contained five mice. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality, and/or increase in mean day to death determined through 21 days.