Screening with a shell vial assay for antiviral activity against cytomegalovirus

An assay based on the shell vial cell culture (SVA) was devised to assess differences in fluorescence patterns of 8 cytomegalovirus (CMV) strains incubated in the presence or absence of three antiviral substances. The drug concentrations selected for in vitro testing were in the range of peak serum levels achievable in humans (ganciclovir 20 microM, acyclovir 80 microM, and phosphonoformic acid 300 microM). Four days after inoculation of the shell vials, the cell monolayer was fixed, stained with a mixture of monoclonal antibodies to early and late antigens of CMV, and reacted with antimouse fluorescein-labeled globulin. Twenty microM of ganciclovir in the media inhibited the formation of plaque-like foci and specific cytoplasmic fluorescence of laboratory strain AD169 and four clinical isolates. Two documented resistant strains exhibited partial inhibition at 20 microM, and a clinical isolate exhibited incomplete inhibition in three or four occasions. None of the strains was fully inhibited by 80 microM of acyclovir; however, different degrees of partial inhibition were observed. All isolates exhibited high-grade inhibition by 300 microM of phosphonoformic acid. The SVA can be used as a simple screening test for anti-CMV substances and as a screening of CMV resistance to ganciclovir prior to quantitative testing by the more complex plaque reduction assay.