The overall objective of this contract is to provide a resource for preclinical in vitro evaluation of new chemical substances against members of the respiratory virus group -- influenza type A, influenza type B, parainfluenza virus type 3, respiratory syncytial virus, measles, and adenovirus type 5. In the initial studies, experimental compounds will be screened using the CPE inhibition assay determined both by microscopic observation and by colorimetric changes following neutral red uptake. Initial cytotoxicity will be determined by visual inspection of treated cells and by the neutral red assay. Substances that have a selectivity index of ten or greater will be confirmed using the virus yield reduction assay. Cytotoxicity in confirming experiments will use viable cell count determined by Coulter counter. These confirmatory tests are designed to select substances that have potential for further evaluation in animal models and clinical trials. Further testing of active substances will be performed to determine the level of cell toxicity (i.e., DNA, RNA, and protein synthesis inhibition) and the mechanism of action (i.e., time of drug addition, metabolite challenge). When results warrant, additional studies such as testing the substances in combination with other compounds for toxicity and antiviral efficacy, and virucidal studies.
