Article Figures & Data
Figures
- FIG. 1.
Cytotoxicity of zinc salts for HEp-2 cell monolayer cultures. Cytotoxicity was determined by the addition of zinc salts to 90% confluent cell monolayers in a 24-well plate and by incubation at 37°C for 96 h. At the end of the incubation period, cells were trypsinized and resuspended in medium and cell viability was assessed with trypan blue exclusion. Cell viability was determined by comparing zinc to mock-treated cell monolayers. Values represent mean and SEM results from six independent experiments.
- FIG. 2.
Effect of zinc salts on RSV yield when present during adsorption, penetration, and egress. Virus was combined with various concentrations of zinc salt or control salts in 50 mM MOPS-buffered Opti-MEM I medium, pH 7.2, in a total volume of 250 μl, and the mixture was incubated at 37°C for 2 h. Control wells without zinc (0 mM) were treated similarly. At the end of the incubation period, the zinc-virus mixtures were inoculated onto HEp-2 cell monolayer cultures. After 96 h supernatants were collected and the viral titer was determined by standard 4-day plaque assay on HEp-2 cell monolayer cultures. Virus titer values indicate the mean values and SEM of three independent experiments.
- FIG. 3.
Effect of zinc salts on RSV plaque formation when present during adsorption, penetration, and egress. Plaque counts in the presence of indicated salts in a plaque reduction assay with approximately 90 PFU of wild-type RSV strain A2. RSV was incubated with various concentrations of zinc or control salts for 2 h before infection of HEp-2 cell monolayer cultures. The indicated salts were present during adsorption and were added to the overlay placed after adsorption. Percent plaque count was determined by adjusting the plaque count for the mock-treated cultures to 100. Values indicate the mean values and SEM of three independent experiments. There was a significant difference in inhibition of RSV in zinc treatment experiments compared to those including treatment with calcium, magnesium, or manganese salts (P < 0.001). There was not a significant difference in percent inhibition between the zinc salts.
- FIG. 4.
Effect of zinc salts on RSV yield when present only during virus penetration. HEp-2 cell monolayer cultures were prewashed with ice-cold PBS, infected with RSV at an MOI of 0.1 PFU/ml, and incubated at 4°C for 2 h. Nonabsorbed virus was removed by repeated washing three times with cold PBS; medium containing zinc or control salt was added, and further incubation at 37°C for 4 h took place. Excess salt was removed and replaced with Opti-MEM medium. After 96 h of incubation at 37°C, virus was harvested and titers were determined. Virus titers represent means for three independent experiments.
- FIG. 5.
Effect of zinc salts on RSV yield when added only after absorption and/or penetration. HEp-2 cell monolayers were infected with RSV at an MOI of 0.1 PFU/cell and were then allowed to adsorb for 1 h at 37°C. Nonadsorbed virus was removed by washing with PBS three times, and the medium was replaced with medium containing the desired concentration of zinc or control salt. After 96 h, virus was harvested from the cultures and frozen at −70°C pending determination of viral titer. Viral titers represent mean values from three independent experiments. The mean viral yield for the mock-treated culture was 4.7 log 10 PFU/ml (SEM, 0.4 log 10 PFU/ml). The effect of zinc salt on viral yield was concentration dependent and was significantly different from that found with mock-treated and the control salts. A statistically significant difference in viral yield was not observed between the zinc salts.
- FIG. 6.
Effect of zinc salts on RSV plaque formation when added only after adsorption and/or penetration. RSV plaque counts in the presence of the indicated salts in a plaque reduction assay with approximately 90 PFU of wild-type RSV strain A2. HEp-2 cell monolayers were infected with virus, incubated at 37°C for 1 h, and then overlaid with methylcellulose containing various concentrations of zinc or control salts. Percent plaque counts were determined by adjusting the plaque count percentage for the mock-treated cultures to 100. Values are means and SEM of two independent experiments.
- FIG. 7.
Inhibitory effect of zinc salts on RSV plaque formation when the cell culture monolayer was pretreated with zinc salts. RSV plaque counts in the cell cultures pretreated with indicated salts in a plaque reduction assay with approximately 90 PFU of wild-type RSV strain A2. Percent plaque count was determined by adjusting the plaque count percentage for the mock-treated cultures to 100. Values are means and SEM of three independent experiments.
