FACS ANALYSIS OF ANTIVIRAL DRUGS ON HCMV REPLICATION

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DESCRIPTION (Adapted from applicant's abstract): The long term goals of this project are 1) to validate a flow cytometry drug susceptibility assay for use with antiviral compounds effective against human cytomegalovirus; and 2) to demonstrate the feasibility of using fluoroochrome labeled monoclonal antibodies to viral antigens and flow cytometry to begin to determine the mode of action of these antiviral compounds. Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in immunocompromised patients. There are three licensed antiviral drugs for use for treatment of HCMV associated retinitis in this patient population and a number of novel antiviral compounds are under development. The current procedures for determining drug susceptibility of HCMV clinical isolates are time consuming, labor intensive, and subjective. Mode of action studies are also labor intensive and time consuming. There is a need for better methods for measuring the susceptibility of HCMV chemical isolates to antiviral drugs and their mode(s) of action. Novel antiviral drugs effective against HCMV infection have been obtained from pharmaceutical companies. The susceptibilities of drug sensitive and drug resistant HCMV laboratory strains and clinical isolates to these antiviral drugs will be assessed using this flow cytometry drug susceptibility assay. Fluorochrome labeled monoclonal antibodies to immediate early (IE), early and late HCMV antigens, the DNA stain propidium iodide , and flow cytometry also will be used to begin to elucidate the mode(s) of action of these novel antiviral compounds. The introduction of flow cytometry technology for use in antiviral drug research will speed the discovery of more effective and safer drugs effective against HCMV. Extension of this flow cytometry technology for drug susceptibility and mode of action studies to antiviral drugs effective against other viruses will eventually make this a broadly used technique in antiviral research.