Antiviral effect of mefloquine on feline calicivirus in vitro

Abstract

Feline calicivirus (FCV) is an important viral pathogen of domestic cats causing clinical signs ranging from mild to severe oral ulceration or upper respiratory tract disease through to a severe fatal systemic disease. Current therapeutic options are limited, with no direct acting antivirals available for treatment. This study screened a panel of 19 compounds for potential antiviral activity against FCV strain F9 and recent field isolates in vitro. Using a resazurin-based cytopathic effect (CPE) inhibition assay, mefloquine demonstrated a marked inhibitory effect on FCV induced CPE, albeit with a relatively low selectivity index. Orthogonal assays confirmed inhibition of CPE was associated with a significant reduction in viral replication. Mefloquine exhibited a strong inhibitory effect against a panel of seven recent FCV isolates from Australia, with calculated IC50 values for the field isolates approximately 50% lower than against the reference strain FCV F9. In vitro combination therapy with recombinant feline interferon-ω, a biological response modifier currently registered for the treatment of FCV, demonstrated additive effects with a concurrent reduction in the IC50 of mefloquine. These results are the first report of antiviral effects of mefloquine against a calicivirus and support further in vitro and in vivo evaluation of this compound as an antiviral therapeutic for FCV.

Keywords: Antiviral; Cats; Feline calicivirus; Mefloquine.

Fig. 1

Results of FCV antiviral screening experiment. Cells were pre-treated with various compounds for 1 h prior to infection with FCV F9 at MOI 0.01. Antiviral efficacy was determined 48 hpi using the resazurin-based CPE inhibition assay (b). A concurrent cytotoxicity screen was performed using the same protocol with the exception that cells were mock infected (a). Each treatment was performed in triplicate and repeated in three independent experiments. Results represent Mean ± SE. ATA, aurintricarboxylic acid; HMA, hexamethylene amiloride. Red dotted line = 75% inhibition of CPE.

Fig. 2

Titration of mefloquine against FCV using the resazurin-based CPE inhibition assay. Each treatment was performed in triplicate and repeated in three independent experiments. Results represent Mean ± SE. Calculated IC50, CC50, and SI values are shown in the accompanying table.

Fig. 3

Virus yield reduction assay for mefloquine against FCV. Cells were pre-treated with various dilutions of mefloquine for 1 h prior to infection with FCV F9 at MOI 0.05. Extracellular virus titre was calculated at 12 (blue circles) and 24 hpi (red squares) with a TCID50 end point assay. Titre of untreated control is defined as 100%. Each treatment was performed in triplicate and repeated in two independent experiments. Data represent Mean ± SE. Calculated IC50 and SI values for each time point are shown in the accompanying table.

Fig. 4

Antiviral efficacy of mefloquine against recent Australian field isolates of FCV. Antiviral efficacy was determined 48 hpi using the resazurin-based CPE inhibition assay. Each treatment was performed in triplicate and repeated in three independent experiments. Results represent Mean ± SE.

Fig. 5

Antiviral titration of mefloquine against Australian field isolates of FCV. Antiviral efficacy was determined 48 hpi using the resazurin-based CPE inhibition assay. Each treatment was performed in triplicate and repeated in three independent experiments. Results represent Mean ± SE.

Fig. 6

Effect of combination treatment with mefloquine and rFeINF-ω against FCV F9. Cells were pre-treated with varying combinations of rFeINF-ω and mefloquine prior to infection with FCV F9 at MOI 0.01. Antiviral efficacy was determined 48 hpi using the resazurin-based CPE inhibition assay. Each treatment was performed in triplicate and repeated in three independent experiments. Results represent Mean ± SE. IC50 values for mefloquine were calculated for each rFeINF-ω concentration.