Cytotoxicity test of copper chloride on F81 cells
The results of the cytotoxicity assay showed that the relative cell viability was above 85% after treatment with copper chloride at a concentration of 20~80 μM for 24 h or 72 h. When the concentration of the copper chloride was less than or equal to 200 μM (below CC50), the relative cell viability of the F81 cells was higher than 50% after 24 h or 72 h of treatment, indicating that copper chloride can be regarded as non-toxic in this concentration range. Therefore, a concentration of 200 μM copper chloride was used as the maximum concentration for the antiviral experiments.
Fig. 1 Cytotoxicity assay of copper chloride to F81 cells. A CCK-8 assay was used to measured cytotoxicity to F81 cells exposed to copper chloride at concentrations of 400, 200, 100, 80, 60, 40, and 20 µM during incubation at 37 °C and 5% CO2 for 24 h or 72 h. The relative activity of 0.4% DMSO-treated F81 cells was considered to be 100%, and the cytotoxicity was shown as the percentage of cell activity with respect to the DMSO mimetic treatment. Each value represents three independent replicate experiments.
Antiviral effect of copper chloride at different doses
To evaluate the antiviral effects of different doses of copper chloride against FCV, we examined the virus titre and RNA levels of FCV in the F81 cells treated with different doses of copper chloride. The results showed that the virus titre was significantly lower than that of the mock group after the cells were exposed to 60, 80, 100, and 200 μM concentrations of copper chloride (p < 0.001) (Fig. 2A). The relative RNA levels of the FCV were significantly reduced compared to the mock group when the copper chloride concentration was 80 or 100 μM (p < 0.005), and the decrease was highly significant at 200 μM (p < 0.001) (Fig. 2B). In addition, all the results indicated that the antiviral effect of copper chloride on the F81 cells was dose-dependent. The IC50 of the copper chloride to FCV was determined to be 5.1 μM (Fig. 2C).
Fig. 2 The antiviral effect of different doses of copper chloride (20-200 µM) on the 100 TCID50 FCV-infected F81 cells. After incubation for 28 h at 37 ℃ and 5% CO2, the virus titre (A) and the relative RNA levels (B) of the FCV were detected. (C) The IC50 of the copper chloride to the FCV was determined; * p <0.0332; ** p <0.0021; *** p <0.0002; and **** p <0.0001.
Indirect immunofluorescence assay
To further evaluate whether the antiviral effect of copper chloride against FCV was dose-dependent, we performed an indirect immunofluorescence assay (IFA). The results showed that intense green fluorescence signal was observed in the 0 μM group, and only weak fluorescence signals were observed at the concentrations of 60, 40, and 20 μM, indicating a dose-dependent relationship after greyscale scanning (Fig. 3B). There was almost no fluorescence signal at the concentrations of 80, 100, and 200 μM or for the control group compared to the mock group (Fig. 3A). In addition, the F81 cells treated with 200 μM copper chloride showed slight rounding and polymerization. This finding also validates the range of non-toxic concentrations of copper chloride previously described.
Fig. 3 IFA verified the antiviral effect of copper chloride. (A) F81 cells were infected with different concentrations of copper chloride (20-200 μM) and FCV (100 TCID50), and a mock treatment group containing 0.4% DMSO and a negative control group uninfected with FCV were used as controls. After incubation for 1 h at 37°C and 5% CO2, IFA of the F81 cells was performed. (B) Two experimental wells were selected in the 96-well cell plate, and fluorescence signals were scanned in randomly selected areas in each well using Image J software. The experimental result was expressed as the average of two fluorescence signals. The antiviral effect of the copper chloride was evaluated by scanning the fluorescence signal; **** p <0.0001.
Antiviral effect of copper chloride for different treatment times
To evaluate the antiviral effect of copper chloride to FCV at different treatment times, we examined the virus titer and RNA levels of FCV in F81 cells at different treatment times. The experimental data showed that 80 μM copper chloride had good antiviral effect on FCV and lower cytotoxicity. Therefore, we selected this concentration of copper chloride for subsequent experiments. The results showed that the virus titre and the relative RNA level of the FCV were significantly lower in the copper chloride treatment group after -1, 0, and 1 h of FCV infection compared with that of the mock group (p < 0.001), and there was also a significant difference after 4 and 8 h of infection (p < 0.002). After 16 h of virus infection, there was no significant difference between the virus titre or relative RNA level of the FCV and the mock group, indicating that the antiviral effect of copper chloride (80 μM) was not significant after 16 h of FCV infection (Fig. 4A and B). The RT-qPCR data also obtained the similar results. Thus, the antiviral effect of copper chloride on FCV was mainly in the early stages of virus proliferation.
Fig. 4 The antiviral effect of copper chloride on FCV at different time points. The F81 cells were infected with 100 TCID50 FCV and then treated with 80 µM copper chloride for -1, 0, 1, 2, 4, 8, and 16 h. After incubation for 24 h at 37 °C and 5% CO2, the virus titre (A) and relative RNA levels (B) of the FCV were detected. NS, p > 0.1234; ** p <0.0021; *** p <0.0002; and **** p <0.0001. ( ‘-1 h’ indicated that the cells were treated with CuCl2 1 h before infection)
Antiviral effect of copper chloride on different strains
For other strains of FCV, the antiviral effects of copper chloride were validated. As previously described, we determined the viral titre and relative viral RNA levels of the different strains after copper chloride treatment. The results showed that 80 μM copper chloride used to treat the other strains of FCV (CH-JL1, CH-JL3, CH-JL4, and CH-SH) significantly reduced the viral titre and relative viral RNA levels of the infected F81 cells (Fig. 5A and B). These results indicated that copper chloride had a strong antiviral effect against strains of FCV with differing genotypes.
Fig. 5 The antiviral effect of copper chloride for different FCV strains. The F81 cells were infected with different FCV strains at 100 TCID50 and treated with 80 µM copper chloride. After incubation for 28 h at 37 °C and 5% CO2, the virus titre (A) and the relative RNA levels (B) of the FCV were detected; * p < 0.1234 and ** p < 0.0021.
Synergistic effect of ribavirin and the antagonistic effect of F(ab')2
The checkerboard method was used to mix different compounds, and the antiviral effect of the compound combination was evaluated using SynergyFinder. The results showed that the combination of copper chloride and ribavirin had a synergistic effect on the protection of FCV-infected F81 cells within a specific concentration range (Fig. 6A). The ZIP model was used to calculate the average score for the synergy of the two compounds, and it was found to be 9.687 (Fig. 6B). Conversely, the combined use of copper chloride and F(ab')2 showed antagonism, and the average score of the two compounds calculated by the ZIP model was -20.798 (Fig. 6C and D).
Fig. 6 The antiviral effect of the compound combination. (A and C) The compounds were diluted to the indicated concentrations and used in combination to treat FCV infection. The results of the RT-qPCR were statistically analyzed by the methods described above, and the effects of the drug combination were evaluated using SynergyFinder. (B and D) Different concentrations of compound interaction scores were calculated using the ZIP model. The synergy score for the ZIP model was expressed as the average of all δ scores in the dose-response landscape, and the red portion of the graph indicates synergy. All experiments were repeated three times.