Viruses, cells and compounds
The FCV CH-SH strains and F81 cells were provided by the Institute of Military Veterinary Medicine, Academy of Military Medical Science. FCV CH-JL1/CH-JL2/CH-JL3/CH-JL4 were isolated and stored in our laboratory [38]. The horse anti-FCV immunoglobulin F(ab')2 was produced and stored in our laboratory [39]. The copper chloride dihydrate (Aladdin, China) product number was C111685. The ribavirin (Aladdin, China) product number was R101754.
Cytotoxicity assay for copper chloride
Copper chloride was diluted in DMSO to a concentration of 100 mM as a stock solution, and the stock solution was diluted to different concentrations (400 μM, 200 μM, 100 μM, 80 μM, 60 μM, 40 μM, and 20 μM) with MEM containing 2% FBA. The cells were incubated at 37 ℃ in 5% CO2 for 24 h or 72 h. After washing the cells twice with PBS solution, 180 μL of FBA-free MEM and 20 μl of CCK-8 reagent (CCK-8; Dojindo, Japan) were added to each well. After incubation at 37 ℃ for 1-2 h, the optical density (OD) was determined at 450 nm using a Cmax Plus microplate reader (Molecular Devices, USA). The cell viability was calculated by the formula [OD450 (compound) - OD450 (blank)]/[OD450 (control) - OD450 (blank)] × 100%. The copper chloride concentrations that were less than 50% of the cytostatic concentration (CC50) were defined as nontoxic [40].
Virus titre and genome detection
The virus solution to be detected was diluted serially 10-fold. Each concentration solution was added to each column at 100 μL per well, which was repeated 8 times for each concentration, followed by the addition of 100 μL of 2% MEM. The control was established in virus-free medium and cultured at 37 °C in 5% CO2. The virus TCID50 was calculated using the Reed and Muench formula. Relative RT-qPCR was used to evaluate FCV gene expression. Briefly, first, the RNA of the virus was extracted from the mixture of cells and medium (by freezing and thawing three times) with the Simply P Total RNA Extraction Kit (Bioflux, China); then, the RNA was reverse-transcribed into complementary DNA (cDNA) (Thermo, USA), followed by RT-qPCR using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, China). The RT-qPCR conditions were based on the Applied Biosystems 7500 Fast Real-Time PCR System with TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, China). The upstream and downstream sequences of the FCV primers were 5′-GCAGGTTGGGATAAACATGGA-3′ and 5′-CACGAGGCGATTGAGTTGAG-3′, and for GAPDH, the sequences were 5’-TGGAAAGCCCATCACCATC-3’ and 5’-ACTCCACAACATACTCAGCACCA-3’.
Antiviral effect of copper chloride on FCV
F81 cells at 100% confluence were infected with FCV CH-JL2 at 100 TCID50, while 200 μL of different concentrations of copper chloride (200-20 μM) were added to the medium. MEM containing 0.4% DMSO was used for the mock treatment group. After incubation for 28 h at 37 °C in 5% CO2, the TCID50 levels of the virus and the RT-qPCR results were determined to assess the antiviral effect on FCV. The half-maximal inhibitory concentration (IC50) of copper chloride for FCV was also determined, and the results were plotted using GraphPad Prism 8.
Indirect immunofluorescence assay (IFA)
To more intuitively observe the antiviral effect of copper chloride on FCV, we performed an IFA [39]. As described above, a monolayer of F81 cells infected with 100 TCID50 of FCV was exposed to different concentrations of copper chloride and then fixed in 80% cold acetone for 30 min. Next, the cells were washed 5 times with PBS containing 0.05% Tween-20 (PBST). Then, primary antibody (VMRD, USA) diluted 300-fold with 1% BSA was added, and the cells were incubated for 1 h at 37 °C. The supernatant was discarded, the cells were washed 5 times with PBST, and 200-fold diluted FITC-labelled rabbit anti-cat secondary antibody (Bioss Antibody, China) was added to the cells in the dark and incubated at 37 °C for 1 h. The fluorescence was observed under an inverted Leica DMi8 fluorescence microscope (Leica, Germany), and the fluorescence results were analysed by greyscale scanning.
Effect of different copper chloride treatment times on FCV
To determine whether the antiviral effect of copper chloride on F81 cells was time-dependent, copper chloride solution at a final concentration of 80 μM was administered -1 h, 0 h, 2 h, 4 h, 6 h, 8 h, and 16 h after FCV infection. MEM containing 0.4% DMSO was used for the mock treatment group. After incubation for 24 h at 37 °C in 5% CO2, the TCID50 level was determined, and the RT-qPCR results were assessed to determine the amount of virus.
Antiviral effect of copper chloride on different strains
To determine whether copper chloride also has antiviral effects on other FCV strains, we diluted different strains of FCV (CH-JL1, CH-JL3, CH-JL4, and CH-SH) to 100 TCID50 and infected F81 cells, which were subsequently treated with copper chloride solution at a final concentration of 80 μM. After incubating the cells with virus for 28 h at 37 °C in 5% CO2, the TCID50 level was determined, and the RT-qPCR results were assessed to determine the amount of virus.
Combination of copper chloride and ribavirin or F(ab')2
To evaluate the combined action of the compounds, we diluted the different drugs by the checkerboard method and mixed the two drugs thoroughly to determine the combined effect on FCV of different concentrations of copper chloride (0–40 μM) and ribavirin (0–15 μM)/F(ab')2 (0–300 μg). The results from the RT-qPCR experiments were statistically analysed in a manner described previously, and the effects of the drug combinations were evaluated using SynergyFinder [41]. The zero-interaction efficiency (ZIP) model [26] was used to calculate the mutual scores of the different concentrations of the drug. For each treatment, at least three data sets from triplicate independent experiments were analysed. The results are expressed as the mean and standard error of the mean (SEM).
Statistical methods
All experiments were performed three times independently in triplicate. The data are expressed as the mean ± standard deviation (SD). The significance of the differences between the groups were determined by paired t-tests and one-way/two-way analysis of variance.