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In the first experiment two rhesus (Macaca mulatta) and two cynomolgus (Macaca fasicularis) monkeys were inoculated intranasally (i.n.) with 3 × 107 plaque-forming units (PFU) of the Peking strain of Japanese encephalitis virus (JEV) to establish the time course of infection and resulting mortality. The onset of clinical signs for both species of monkeys occurred on days 5 to 9, with fever of several days duration, anorexia and depression. Death ensued in 11 to 12 days. An i.n. median lethal dose equivalent to 2.5 × 104 PFU of the Peking strain of JEV was determined in 16 additional cynomolgus monkeys. Clinical signs of infection, virus-neutralizing antibody formation, and mortality were dose-dependent for the doses of virus inoculated. Total peripheral blood leukocyte and neutrophil values increased midway during the course of infection in monkeys that died with encephalitis. Microscopic lesions of JE were similar in monkeys that died following virus challenge. No species-related differences in response to JEV challenge were evident. A nuclease-resistant complex of polyriboinosinic·polyribocytidylic acid, poly-l-lysine and carboxymethylcellulose [poly(ICLC)] reduced mortality by 50% in monkeys treated initially 8 or 24 h after virus challenge. Mean survival time of nonsurvivors was prolonged 3.5 days and microscopic lesions of encephalitis were less severe in the poly(ICLC)-treated monkeys when compared to infected-untreated monkeys. The response of rhesus and cynomolgus monkeys to JEV challenge by the i.n. route of inoculation thus provides a useful model for the study of potential antiviral compounds in host defense against Japanese encephalitis.