In vitro antiviral effects of GS-441524 and itraconazole combination against feline infectious peritonitis virus

Abstract

Feline infectious peritonitis virus (FIPV: virulent feline coronavirus) causes a fatal disease called feline infectious peritonitis (FIP) in wild and domestic cat species. Recent studies identified several antiviral drugs that are effective against FIPV. Drug combination is one of the important strategies in the development of novel treatments for viral infections. GS-441524, a nucleoside analog, and itraconazole, a triazole antifungal drug, have been reported that have antiviral effect against FIPV. This study aims to investigate whether the combination of GS-441524 and itraconazole has synergic antiviral effect against FIPV. The antiviral effect was measured by plaque reduction assay using felis catus whole fatus-4 cell. The plaque reduction of GS-441524 against type I FIPVs increased as the concentration of itraconazole increased. The similar result was obtained for type II FIPV. In addition, the calculated combination index (CI) demonstrated that there was a strong synergy between GS-441524 and itraconazole. It is concluded that the combination of GS-441524 and itraconazole may enhance the individual effect of each drug against replication of type I FIPVs and may contribute to development more effective treatment strategy for FIP.

Keywords: Coronavirus; Drug combination; Feline infectious peritonitis virus.

Fig. 1

Cytotoxic effects of GS-ICZ combination on Felis catus whole fetus-4 cells (fcwf-4 cells). The fcwf-4 cells were seeded on 96-well plates. The cells were treated with ICZ for 24 h at the indicated concentrations. The cells were washed and the serial diluted GS-441524 were added to the wells. After incubation for 24 h, the culture supernatants were removed. WST-8 solution was added, and the cells were incubated for 1 h. The absorbance of formazan produced was measured at 450 nm. Percentage cytotoxicity was calculated using the following formula: Cytotoxicity (%) = [(OD of compound-untreated cells−compound-treated cells)/(OD of compound-untreated cells)] × 100. The results are shown as mean ± SE. Data represent three independent experience.

Fig. 2

The effects of GS-ICZ combination on type I FIPV KU-2. The fcwf-4 cells were pretreated with ICZ for 24 h. After viral adsorption, the cells were cultured in CMC-MEM containing GS-441524. The cells were fixed and stained with 1% crystal violet and the resulting plaques were then counted. The plaque reduction percentage was calculated using the following formula: Plaque reduction percentage (%) = [(plaque number of compound-treated cells)/(plaque number of compound-untreated cells)] × 100. (A) Plaque reduction in treated with GS-441524 and ICZ. The results are shown as mean ± SE. Data represent three independent experience. (B) Crystal violet stain of FIPV infected fcwf-4 cells treated with GS-441524 and ICZ. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The effects of GS-ICZ combination on type I and II FIPV. The fcwf-4 cells were pretreated with ICZ for 24 h. After viral adsorption, the cells were cultured in CMC-MEM containing GS-441524. The cells were fixed and stained with 1% crystal violet and the resulting plaques were then counted. The plaque reduction percentage was calculated using the following formula: Plaque reduction percentage (%) = [(plaque number of compound-treated cells)/(plaque number of compound-untreated cells)] × 100. The results are shown as mean ± SE. Data represent three independent experience. (A) type I FIPV Black. (B) type II FIPV 79–1146. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

FI-CI plots of GS-ICZ combination. The X-axis and Y-axis indicate fractional inhibition and combination index, respectively. (A) type I FIPV KU-2. (B) type I FIPV Black.