application): The proposed studies in this application represent a collaborative research endeavor focused on employing a consensus viral susceptibility assay for human cytomegalovirus (HCMV) clinical isolates and measures of HCMV burden in body fluids to determine the role of resistance and viral load in clinical failure of antiviral therapy in the treatment of HCMV disease. Furthermore, the consensus plaque assay developed by the applicants will be expended for use on clinical HCMV isolates and resistance will be confirmed by nucleic acid sequence analysis and marker transfer. Susceptibility will be determined on clinical isolates from patients enrolled in trials of ganciclovir, foscarnet, and cidofovir. The use of flow cytometry to perform phenotypic assays for susceptibility of clinical isolates will also be used and the results obtained will be compared with those from the plaque reduction assays. The HCMV burden in blood, vitreous fluid, and urine will be measured by polymerase chain reaction (PCR) for HCMV DNA and antigen detection. Finally, the use of flow cytometry on blood specimens will be developed with HCMV monoclonal antibodies for measuring HCMV infection which can be used to monitor HCMV viral burden and the effect of antiviral therapy on viral burden.
