Four methods for analyzing viral susceptibility to antiviral substances were compared. In two methods viral products were measured: late viral proteins were measured by an enzyme-linked immunosorbent assay and viral DNA was measured by DNA hybridization. Infectious virus was quantified in the other two assays as the number of plaques and the yield of virus. The enzyme-linked immunosorbent assay procedure in our hands detected the smallest amounts (lowest proportions) of thymidine kinase-deficient herpes simplex virus type 1 mixed with wild-type virus. The thymidine kinase-deficient proportion of the herpes simplex virus type 1 isolate increased rapidly in the presence of acyclovir in cell culture.Several methods for analyzing viral drug susceptibility have been reported. All assays require incubation for some time in cell culture in the presence of the antiviral substance. Subsequently, the virus is quantified. The number of infectious virus particles can be quantified by plaque counting (4,11) or by viral yield after a secondary cell culture passage of virus followed by plaque enumeration (21,22). Recently, the plaque assay was refined by incorporating a radioactively labeled substrate into cells with a competent viral thymidine kinase (TK) (14, 26). The virally induced cytopathic effect can be measured by dye uptake (16, 17). Furthermore, virus-specific products such as viral DNA or protein production can be quantified (7,24,25).The object of this study was to compare the sensitivities of different methods. Therefore, the smallest amount of a known drug-resistant virus that could be detected in the presence of drug-susceptible virus was quantified in several ways. A wild-type herpes simplex virus type 1 (HSV-1) strain and a TK-deficient (TKD) HSV-1 strain lacking thymidine phosphorylation properties were mixed in various proportions and subsequently analyzed by four methods for estimation of viral drug susceptibility.
MATERIALS AND METHODSViruses. C42 is a HSV-1 laboratory strain known to induce large amounts of TK (8). C915 is a true TKD variant virus strain originally obtained from a patient with 5-iodo-2'-deoxyuridine-treated HSV-1 conjunctivitis (9). C915 showed decreased susceptibility toward all TK-dependent drugs tested. Both strains were originally supplied by S. Gronowitz and C. Kallander, Biomedical Center, Uppsala, Sweden.Cells. All experiments were performed with human embryonic lung (HL) fibroblasts. Such cells are particularly suitable for herpes simplex virus susceptibility testing of antiviral drugs since they are primary diploid cells of human origin with a finite life span and contain only low amounts of thymidine (10). ELISA. After 24, 48, or 72 h of incubation, the cells were scraped off duplicate wells and lysed, and the viral antigen was quantified by using a sandwich enzyme-linked immunosorbent assay (ELISA) (24,25). The interassay coefficient of variation (CV) for the method was 6.7%.Plaque assay. After the adsorption period, the inoculum was removed, and medium supplemented with 0.5% g...
