Antiviral activity of itraconazole against type I feline coronavirus infection
Feline coronaviruses (FCoVs) are the causative agents of severe systemic disease (feline infectious peritonitis: FIP) in domestic and wild cats. FCoVs have been classified into serotypes I and II. Type I FCoV is the dominant serotype (approximately 70–90%) worldwide. Therefore, it is necessary to provide antiviral agents for type I FCoV infection. In this study, we demonstrated that itraconazole (ICZ), practically used for fungal infections in cats, inhibits the type I FCoV infection. ICZ also exhibited antiviral effect in cells after viral infection, suggesting that ICZ could potentially be used as a therapeutic.
feline infectious peritonitis
feline enteric coronavirus
Felis catus whole fetus-4
50% cytotoxic concentration
multiplicity of infection
median tissue culture infective dose
Introduction, methods and results
Feline coronavirus (FCoV; family Coronaviridae, genus Alphacoronavirus) is an enveloped, single-stranded, positive-sense RNA virus. FCoV exists as two different biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) . The former causes mild enteritis (usually subclinical infection), and the latter causes the highly lethal systemic disease FIP. Although antiviral drugs and vaccines against FIP have been investigated, no method has been established for practical use . Furthermore, FCoV also exists as two serotypes: type I FCoV (type I FECV and type I FIPV) and type II FCoV (type II FECV and type II FIPV) . Serological and genetic surveys showed that type I FCoV is dominant worldwide [4, 5, 6].
We previously reported that type I FCoV is closely associated with cholesterol throughout the viral life cycle . We also demonstrated that U18666A, the cholesterol transport inhibitor, strongly inhibits type I FCoV infection . Based on these findings, U18666A may be applied as a therapeutic drug for FIP. However, to our knowledge, U18666A is not approved for veterinary practical use. To use U18666A to treat FIP, pharmacokinetic, pharmacodynamic, and safety studies must be performed for cats. As with U18666A, several candidate antiviral drugs targeting type I FCoV have been identified [9, 10]. However, none of drugs scientifically demonstrated to exhibit a therapeutic effect on FIP are practically used. To solve these problems, it is desirable to identify a potent antiviral agent for FCoV infection among drugs generally used for cats.
Itraconazole (ICZ) is classified as an azole antifungal . It has low toxicity and can be used to treat fungal infections in immunocompromised patients . It is commonly used by veterinarians to treat fungal infections in dogs and cats. Recently, ICZ was suggested to be effective for enteroviral infection (poliovirus, rhinovirus, and coxsackievirus) . We previously investigated the antiviral effects of cholesterol transport inhibitors, including U18666A, and confirmed that ICZ inhibits type I FCoV infection . However, the influence of ICZ on FCoV infection has not been investigated in detail. In this study, we examined the antiviral effects of ICZ on FCoV.
The CC50, IC50, and SI of itraconazole
Furthermore, we examined the effects of ICZ on plaque formation after infection of fcwf-4 cells with FIPV-I KU2. The virus at MOI of 0.01 was added to the culture and adsorbed by fcwf-4 cells at 37 °C for 1 h. After washing, cells were cultured in MEM at 37 °C for 1 or 3 h. After exchanging MEM for CMC-MEM containing ICZ, cells were cultured at 37 °C for 48 h. The percentage of plaque inhibition was measured as described above. Post-treatment with ICZ inhibited FIPV-I KU2 plaque formation to a degree comparable with pre-treatment (Figure 2D). In contrast, the percentage of plaque inhibition by FIPV-II 79-1146 was slightly affected by post-treatment with ICZ.
Of the 2 serotypes, ICZ inhibited type I FCoV infection. As approximately 70–90% of cats with FIP are infected with type I FCoV [4, 5, 6], it would be reasonable to test the use ICZ as an anti-FIPV agent. On the other hand, ICZ did not affect type II FCoV, suggesting that the antiviral effects of ICZ differ depending on the serotype of FCoV. The incidence of type II FIPV-induced FIP in Japan and Taiwan is higher than that in Europe [6, 17, 18], and the combination of ICZ with other drugs is necessary in these countries.
A difference was noted between the results of plaque inhibition and virus titration assays in virus-infected cells treated with 2.5 μM ICZ: a significant inhibitory effect on type I FIPV infection was observed in the former but no significant inhibitory effect was noted in the latter. Since plaque inhibition assay measures the infectivity titer of parental virus, whereas virus titration assay measures that of progeny virus, these findings suggest that 2.5 μM ICZ strongly inhibited parental virus infection but the concentration was insufficient to inhibit progeny virus production. However, the titer of produced progeny virus and results of plaque inhibition assay strongly suggest that progeny virus infection is inhibited in cells treated with 2.5 μM ICZ.
Boothe et al.  previously reported the pharmacokinetic variables of ICZ in cats. According to their report, the peak blood concentration (Cmax) was 1.1 ± 3.6 μg/mL (1.6 ± 5.1 μM) in cats treated with oral ICZ at 10 mg/kg once, and it reached 3.4 ± 1.3 μg/mL (4.8 ± 1.8 μM) in cats treated with oral ICZ twice within a 12-h interval. In the present study, plaque formation and viral antigen expression were inhibited in fcwf-4 cells treated with 2.5 μM ICZ, and similar effects were noted in cells treated with ICZ after infection with FIPV-I KU2, suggesting that administration of ICZ at 10 mg/kg twice daily to cats diagnosed with FIP may decrease the viral load. For the treatment of fungal infection in cats, ICZ is administered at a high dose (up to 26.7 mg/kg) in the early phase . Administration of ICZ at a high dose in the early phase may also exert antiviral effects against FIP. Blood alanine aminotransferase has been reported to increase when high-dose ICZ is continued, but this symptom improved after discontinuing drug administration . We recommend that veterinarians use ICZ for treatment of FIP following the fungal infection treatment protocol in cats.
We previously reported that U18666A inhibits cholesterol transport and type I FIPV infection by acting on a cholesterol transporter, Niemann-Pick C1 protein (NPC1) . In this study, inhibition of intracellular cholesterol transport by ICZ was confirmed. Accumulation of cholesterol in the cytoplasm induced by ICZ has been reported by several studies [14, 21] other than the present study, but the mechanism has not been clarified. It has recently been reported that ICZ induces cholesterol accumulation in lysosome by acting on NPC1. Based on this report, ICZ may inhibit type I FCoV replication via the same mechanism as U18666A. In addition, in viruses other than type I FCoV, ICZ inhibits infection by acting on proteins other than NPC1. For example, it has been reported that ICZ acts on oxysterol-binding protein (OSBP) and inhibits formation of the viral replication organelle in enteroviruses . The site of replication of Coronavirus is presumed to be associated with endoplasmic reticulum (ER)-derived structures often referred to as double membrane vesicles (DMVs) , but it is unclear whether OSBP is involved in the formation of the viral replication organelle in coronavirus. The relationship between the infection of type I FIPV and OSBP needs to be investigated.
In this study, we confirmed that ICZ inhibits infection by type I FIPV, the dominant strain in the field, suggesting that ICZ can be applied as a therapeutic drug for FIP. FIP is a “multi-causal disease” involving various risk factors (virulence of FCoV, the status of immunity in host, and the route of virus infection etc…). Taking this fact into consideration, we are planning to perform a clinical trial of ICZ using cats diagnosed with FIP and investigate combination with other therapeutic drugs for FIP at the same time.
The authors declare that they have no competing interests.
TT and TH designed the study. TT and MA performed the experiments. TT, MA, TD, and TH analyzed the data. TT, TD, and TH helped data interpretation. TT and MA wrote the manuscript. TH edited the manuscript and provided funding. All authors read and approved the final manuscript.
We thank Ms. Kumi Satoh for supporting the experiments.
This work was supported by JSPS KAKENHI [Grant-in-Aid for Scientific Research (B)] Grant Number JP16H05039.
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