In vitro antiviral effect of germacrone on feline calicivirus

Abstract

Feline calicivirus (FCV) often causes respiratory tract and oral disease in cats and is a highly contagious virus. Widespread vaccination does not prevent the spread of FCV. Furthermore, the low fidelity of the RNA-dependent RNA polymerase of FCV leads to the emergence of new variants, some of which show increased virulence. Currently, few effective anti-FCV drugs are available. Here, we found that germacrone, one of the main constituents of volatile oil from rhizoma curcuma, was able to effectively reduce the growth of FCV strain F9 in vitro. This compound exhibited a strong anti-FCV effect mainly in the early phase of the viral life cycle. The antiviral effect depended on the concentration of the drug. In addition, germacrone treatment had a significant inhibitory effect against two other reference strains, 2280 and Bolin, and resulted in a significant reduction in the replication of strains WZ-1 and HRB-SS, which were recently isolated in China. This is the first report of antiviral effects of germacrone against a calicivirus, and extensive in vivo research is needed to evaluate this drug as an antiviral therapeutic agent for FCV.

Fig. 1

Cell toxicity assay after germacrone treatment. Cells were pre-exposed to 0.4 % DMSO (mock) or germacrone at concentrations of 20, 40, 60, 80, 100 or 200 μM for 24 h. Cell toxicity was evaluated using a CCK8 assay. The activity of DMSO-treated cells was considered to be 100 %, and cell toxicity was plotted as the percentage viable cells relative to mock-treated cells. The values represent three independent experiments

Fig. 2

Evaluation of antiviral effects of germacrone on FCV growth. (A, B) Cells were treated with germacrone at a concentration of 20-100 μM prior to challenge. After a 1-h treatment with germacrone, cells were inoculated with strain F9 at an MOI of 0.1 TCID₅₀ for one hour in the presence of the drug. The time of germacrone treatment was from one hour before challenge until the end of the experiment. Relative vRNA levels in cells (A) and the virus yields in supernatants from infected cells (B) were determined. (C) CRFK cells were treated with the indicated concentrations of germacrone at 37 °C for one hour or 0.4 % DMSO (mock) and then infected with strain F9 at an MOI of 0.1 TCID₅₀ for one hour. Uninfected cells were used as negative controls. Fluorescence (×40) was observed at 12 hpi

Fig. 3

Evaluation of the effects of germacrone treatment on viral attachment and entry. CRFK cells were treated as described in “Materials and methods”, and vRNA levels in groups exposed to germacrone or 0.4 % DMSO (mock) were analyzed during the virus attachment stage (A) and the virus entry stage (B). The data represent three independent experiments

Fig. 4

Evaluation of the effects of germacrone treatment on viral replication. Cells were inoculated with strain F9 at an MOI of 0.1 TCID₅₀ for one hour. The cells were then exposed to germacrone (60-100 μM) at 37 °C for 12 hours. Relative vRNA levels (A) in the cells and virus yields in the cell supernatants (B) were determined

Fig. 5

The time-dependent effects of germacrone on FCV replication. Cells were inoculated with strain F9 at an MOI of 0.1 TCID₅₀. The cells were treated with germacrone at the indicated time points. Relative vRNA levels (A) in the cells and virus yields in the cell supernatants (B) were determined at 12 hpi; ‘-1 h’ indicates pre-treatment with germacrone for one hour before virus challenge. The data represent three independent experiments

Fig. 6

Inhibitory effects of germacrone on other reference stains and field isolates of FCV. Cells were exposed to 60 μM germacrone for one hour and then inoculated with the indicated FCV strains at an MOI of 0.1 TCID₅₀. The recovered virus yields were determined at 12 hpi. The data represent three independent experiments