Potential Therapeutic Agents for Feline Calicivirus Infection

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 μM and 2.7 μM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 μM and 2.5 μM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.


Introduction
Feline caliciviruses (FCV) are members of the Caliciviridae family (genus Vesivirus) and a major pathogen of cats worldwide. The virus has been associated with vesicular and upper respiratory tract disease, especially in multi-cat environments, such as shelters, colonies, and catteries, where FCV is detected in up to 40% of cats [1][2][3]. FCV infections typically cause a variety of clinical manifestations, such as acute respiratory disease and oral ulceration, with less common symptoms including pneumonia and acute arthritis/limping syndrome [4,5]. More recently, highly contagious virulent strains of FCV have emerged and were linked with severe disease (FCV-associated virulent systemic disease (VSD)) and high mortality rates (up to 50%) [6][7][8]. After the first description of FCV-VSD in 2000, outbreaks have occurred in the USA and Europe, which were associated with genetically distinct virulent FCV strains that have evolved locally [8][9][10][11][12][13]. The severe disease has a marked tropism for endothelial and epithelial cells of the skin and parenchymal organs and adult cats are often more severely affected than kittens [14,15]. 10% (v/v) fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Thermo Fisher, Waltham, MA, USA) and 100 µg/mL streptomycin (Thermo Fisher). Cells were grown at 37 • C with 5% CO 2 . The FCV strain F-9 (VR-782™; GenBank accession number M86379) was purchased from ATCC.

NNIs and NAs
Thirteen antiviral compounds were selected based on the reported in vitro antiviral effects against other caliciviruses [33] and included NNIs, NAs, PIs, and the broad-spectrum nitazoxanide. The stock solutions for all compounds were prepared in 100% dimethyl sulfoxide (DMSO) and aliquoted before storage at −20 • C.

FCV Pro-Pol Cloning, Expression and Purification
The Pro-Pol CDS from FCV Urbana (Genbank accession: L40021) was commercially synthesized in the pOA-RQ vector (Life Technologies, Carlsbad, CA, USA) and then sub-cloned into pET26b (Merck Millipore, Burlington, MA, USA) between BamHI and SalI restriction sites using forward and reverse primers: 5 -AGGTAGGATCCAGTGGATTATAAAGACGATG-3 and 5 -AGGTAGTCGACCACTTCAAACACATCAC-3 to produce pVRL345. For the expression of Pro-Pol containing a C-terminal histidine tag, pVRL345-transformed Escherichia coli BL21 (DE3) (NEB, Ipswich, MA, USA) were grown in Luria-Bertani media (2 L) at 37 • C with 100 µg/mL kanamycin until the OD 600 was~0.6. The culture was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 20 h at 25 • C with shaking and bacteria pelleted by centrifugation. Chemical lysis of the pellet was performed as previously described [34], and lysates were loaded onto Ni 2+ columns (BioRad, Hercules, CA, USA) and purified with an imidazole gradient (10-300 mM) using an AKTA start dual-buffer system (GE Healthcare, Little Chalfont, UK). The equilibration buffer consisted of 50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, 5% glycerol (v/v) and 0.1% Triton X-100 (v/v), and the elution buffer was composed of the equilibration buffer with 300 mM imidazole. The purified protein was concentrated using an Amicon ® Ultra centrifugal filter (10 kDa cut-off, Millipore, Tokyo, Japan) and dialyzed against three buffers with decreasing NaCl concentration (300, 150, or 50 mM NaCl, with 25 mM Tris-HCl, 20% glycerol (v/v); 0.05% Triton X-100 [v/v]). All buffers were prepared at pH 8. The protein concentration was determined using a BCA Protein assay kit (Life Technologies).

Cytotoxicity Study
CRFK cells (3.5 × 10 4 cells/well, 100 µL/well), were seeded into flat-bottom 96-well plates and incubated overnight at 37 • C. The cell monolayers were then treated with increasing concentrations of compounds in triplicate (0.2 µM-100 µM), followed by 48 h in incubation. DMSO (vehicle only, 0.5% (v/v)) was used as a negative control. The cytotoxicity of each compound was measured using the CellTitre-Blue viability assay kit (Promega, Madison, WI, USA) according to the manufacturers' instructions. Fluorescence was measured on a FluoStar Optima microplate reader (BMG Labtech, Ortenberg, Germany) and the half maximal cytotoxic concentrations (CC 50 ) were determined with GraphPad Prism v.7 (La Jolla, CA, USA).

Protease FRET Activity Assay and Antiviral Screening
The amino acid (aa) sequence of the cleavage site between the precursor leader capsid (LC) and the mature capsid protein (VP1) of the FCV genome was synthesized as a fluorogenic substrate peptide Dabcyl-FRLE↓ADDG-Edans (GenScript, Piscataway, NJ, USA) and a stock solution (10 mM) was prepared in 100% DMSO. Protease assays were performed in 384-well plates using a reaction volume of 50 µL containing 50 mM HEPES, pH 7.5, 6 mM DTT, 0.5 mM EDTA, 50% glycerol (v/v), 600 ng of FCV Pro-Pol and the fluorogenic substrate. The initial measurements to determine the Michaelis-Menten constant (K m ) of the substrate were performed using increasing concentrations (0-100 µM) with incubation for 1 h at 37 • C. The influence of increasing NaCl concentration (3-130 mM) on protease activity as also evaluated. Following the determination of the K m , inhibition assays were performed with 50 µM of a substrate with either a PI (0-50 µM) or the vehicle control (0.5% DMSO) with incubation for 30 min at 37 • C. Upon cleavage of the substrate at the site indicated (↓), the quenching of Dabcyl fluorescence by the Edans group is abolished and the fluorescence generated was quantified at an excitation wavelength of 360 nm and an emission of 460 nm on a POLARstar plate reader. IC 50 and K m values were determined using GraphPad Prism v.7.

Inhibition of FCV Plaque Formation in Cell Culture
FCV plaque reduction assays were performed as previously described [36,37]. CRFK monolayers (8 × 10 5 cells/well) in 6-well plates were infected with approximately 80 plaque forming units (pfu) of FCV for 1 h at 37 • C, followed by the addition of semisolid agarose overlays containing different concentrations of compounds. Plates were incubated for 24 h, fixed and stained with crystal violet. Plaque numbers were determined for each drug treatment and the DMSO vehicle control was defined as maximal viral infectivity. To determine whether the combination of nitazoxanide and 2CMC had synergistic, antagonistic or additive effects, the percentage of inhibition of FCV infection was assessed over a dose-response matrix that included four concentrations of nitazoxanide (ranging from 0 to 0.6 µM) and 2CMC (0 to 4 µM). The effects of drug combination were assessed using SynergyFinder [38] and the zero-interaction potency (ZIP) model [39] was used to generate synergy scores from a dose-response matrix. Synergistic or antagonistic effects are shown as peaks above or below the horizontal plane, respectively. At least two independent experiments with triplicate datasets were performed for each treatment, with results presented as the mean with standard error of the mean (SEM).

FCV Genome Reduction Assay Using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
RT-qPCR was used to evaluate the reduction in FCV RNA following antiviral treatment. Briefly, CRFK cells (2 × 10 5 cells/well) in 24-well plates were infected with FCV at the multiplicity of infection (MOI) of 0.0005 for 1 h. Media was then replaced with media containing drug and incubated for a further 24 h. FCV viral RNA was extracted from the cells and supernatant using the QIAmp viral RNA kit (Qiagen, Hilden, Germany). Following this, an 83 bp amplicon of the ORF1 region was generated using iTaq™ Universal SYBR ® Green One-Step Kit (BioRad) as described in Reference [40]. A standard curve was generated using a serially diluted plasmid (containing the 3 end of the FCV ORF1) for genome quantitation. The cycling parameters were 50 • C for 20 min, 95 • C for 5 min and 45 cycles of 95 • C for 10 s and 60 • C for 1 min. All reactions were run in duplicate.

Statistical Analysis
Statistical calculations were performed using the GraphPad Prism v.7 software. Data were analyzed using an unpaired t-test with Welch's correction. All error bars depict standard errors of the mean (SEM), and the level of significance are indicated as: NS, not significant, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

FCV Pro-Pol Expression
We successfully expressed the FCV Pro-Pol polyprotein containing a C-terminal 6-histidine tag in E. coli BL21 cells, under the control of the T7 promoter system. From 2 L of the culture, we purified~3.5 mg of Pro-Pol which appeared at the expected molecular mass (78 kDa) by SDS-PAGE. The presence of the His-tag was confirmed by Western blotting.

RdRp In Vitro Assay
To confirm the RdRp activity of the Pro-Pol dual protein, we tested it using an in vitro fluorescence-based transcription assay, where the dsRNA product was detected with PicoGreen dye [35]. The FCV transcriptional activity increased with increasing concentrations of RdRp (250-1000 ng per reaction) ( Figure 1A). Furthermore, a decrease in RdRp activity was observed with increasing NaCl concentration (3-500 mM) ( Figure 1B). The RdRp activity was reduced by 50% in the presence of 60 mM NaCl, and completely inhibited at 200 mM ( Figure 1B).
Dose-dependent inhibitory response curves (0.1-100 µM) were generated to establish the IC 50 values for quercetagetin (2.8 µM) and PPNDS (2.7 µM) ( Figure 2B,C). The CC 50 of each NNI on CRFK cells was determined using the CellTitre-Blue viability assay (Table 1) (Table 1). In addition, PPNDS and quercetagetin were examined using an FCV plaque reduction assay, with the inhibitory activity calculated after 24 h relative to a mock control (DMSO treatment). At 10 µM, no antiviral activity was observed for both compounds (<10% of plaque formation inhibition) ( Table 1).

FCV Protease In Vitro Assay and Test Compounds
Previous studies have demonstrated that recombinant FCV Pro-Pol exhibits a bifunctional activity of polymerase and protease in vitro [18,55]. Therefore, we also established an in vitro FRET fluorescence-based assay to measure FCV protease activity that cleaves the substrate Dabcyl-FRLE↓ADDG-Edans (corresponding to the LC/VP1 cleavage site) and demonstrated a K m value of 33.5 µM ( Figure 3A). In contrast to FCV RdRp, the protease activity was not inhibited with increasing NaCl concentrations ( Figure 3B). The development of the in vitro FRET assay enabled us to test the inhibitory activity of three previously published PIs: GC376 [29], rupintrivir [53] and chymostatin [52]. Of those, only GC376 exhibited an inhibitory effect against the FCV protease in vitro with a 75% inhibition at 50 µM, and further experiments demonstrated an IC 50 of 18.7 µM ( Figure 3C and Table 1).

2CMC and Nitazoxanide Inhibit FCV Infectivity
Five NA compounds were chosen and tested for their antiviral effects against FCV in the cell culture, including; 2CMC, famciclovir, sofosbuvir, T-705, and 7D2M. All NAs tested have previously shown antiviral effects against several viral families, such as caliciviruses, herpesvirus, paramyxoviruses, orthomyxoviruses, and flaviviruses (Table 1). However, the antiviral efficacy of these compounds against FCV infections has not been evaluated thus far. In addition to these NAs, we also tested the broad-spectrum antimicrobial agent, nitazoxanide, whose mechanism of antiviral action has not been fully elucidated [54]. The dose-response of each compound against FCV was examined using a plaque reduction assay. The compounds 2CMC and nitazoxanide exhibited dose-response inhibition of FCV plaque formation at low micromolar concentrations with EC 50 s of 2.6 µM and 0.6 µM (0.2 µg/mL), respectively ( Figure 4A,B). The compound 2CMC demonstrated CC 50 values of >100 µM, whilst nitazoxanide showed value of 12.7 µM (Table 1), and the therapeutic index values (TI = CC 50 /EC 50 ) determined were of >40 and 21.1, respectively.
We also performed RT-qPCR to quantify the FCV RNA levels after antiviral treatment with different concentrations of nitazoxanide or 2CMC. As shown in Figure 4C, a decrease of dose-dependency in FCV RNA levels was observed after 24 h of treatment for both compounds. Nitazoxanide (2.5 µM) resulted in an 80% reduction of FCV RNA levels, whilst 2CMC (10 µM) reduced the RNA levels by 95% compared to the mock-treated cells ( Figure 4C). The combined inhibitory effects of nitazoxanide (0 to 0.6 µM) and 2CMC (0 to 4 µM) were tested over a range of combinations against FCV in the cell culture using the plaque reduction assay. A dose-response matrix was generated and analyzed for synergy using SynergyFinder. The ZIP mode synergy score is presented as the average of all δ-scores across the dose-response landscape, and the peaks above the plane of 0% synergy in the plot indicate synergism. Nitazoxanide and 2CMC displayed a synergistic antiviral effect against FCV. Data were analyzed using an unpaired t-test. ** p < 0.01; *** p < 0.001; NS, not significant. Duplicate (panels C and D) or triplicate values (panels A and B) from at least two independent experiments are presented, and the mean ± SEM are shown for panels A and C.

Combinational Treatment with Nitazoxanide and 2CMC Showed Synergistic Antiviral Effects
To determine the synergistic effects of nitazoxanide with 2CMC, we performed plaque reduction assays over several combined concentrations ( Figure 4D). The synergistic effect is shown as peaks above the horizontal plane, with ZIP synergy scores varying from 0 to 40. The interaction of both compounds resulted in a moderate synergistic effect (ZIP synergy score of 7.796), with a maximal synergy at concentrations of 0.6:1 µM for nitazoxanide and 2CMC, respectively ( Figure 4D).

Discussion
FCV is a common pathogen of cats and usually associated with acute, mild and self-limiting upper respiratory tract disease, however, more recently highly contagious strains of the virus (FCV-VSD) have been reported in the USA, Europe [11,12,56], and three states of Australia (personal communication, https://au.virbac.com/home/vet-newsletter/main/vet-newsletter/research-update-fcv-vsd.html). With the lack of an effective vaccine and/or antiviral treatment for FCV infection, there is a clear unmet need to identify an effective antiviral agent to improve the management and control of FCV infections.
In the present study, we evaluated 15 different compounds, from four different antiviral classes, using in vitro enzyme-and cell culture-based assays, 13 of which have not previously been evaluated against this virus.
We identified the NA 2CMC (EC 50 = 2.5 µM) and the broad spectrum antimicrobial compound nitazoxanide (EC 50 = 0.6 µM or 0.2 µg/mL) as potent inhibitors of FCV replication ( Figure 4A and Table 1). An NA originally designed for use against HCV, 2CMC is a promising calicivirus antiviral and has previously been tested against human and murine norovirus, with similar results to our current study [57][58][59]. Using in vitro assays, Jin et al. [59] showed that tri-phosphorylated 2CMC inhibited human and murine norovirus RdRp activity with IC 50 s of 2.4 µM and 1.4 µM, respectively. In the same study, 2CMC was tested against the human GI.1 norovirus replicon in the cell culture and demonstrated an EC 50 of 8.2 µM. In related studies, 2CMC was also shown to inhibit the murine norovirus (EC 50~2 µM) and the human norovirus replicon (EC 50~1 8 µM) using cell culture-based assays [47,57]. Recently, using a B-cell culture system, 2CMC also effectively inhibited the human norovirus with an EC 50 of 0.3 µM [60]. Therefore, our results are consistent with the inhibitory data obtained against other caliciviruses as reported in the above cited studies.
Valopicitabine, the prodrug form of 2CMC, was used in pre-clinical studies to treat HCV infections, however, after dose-related gastrointestinal adverse events, the drug has been placed on clinical hold by the US Food and Drug Administration (FDA). Although promising results were obtained here and in other studies [58,60], concerns over adverse side-effects may limit its future clinical use to treat calicivirus infections.
Nitazoxanide is a broad-spectrum antimicrobial compound with activity against anaerobic bacteria, protozoa, and viruses [54]. It is an FDA-approved drug licensed for gastroenteritis caused by the parasites Cryptosporidium parvum and Giardia intestinalis [61,62]. In cell cultures, nitazoxanide has been evaluated against several viruses, showing inhibition in the replication of rotavirus (EC 50 0.5 µg/mL), adenovirus (EC 50 0.2 µg/mL), canine coronavirus (EC 50 1 µg/mL), influenza viruses (EC 50 0.2-1.5 µg/mL), among others [54,63]. In the present study, nitazoxanide demonstrated an EC 50 of 0.2 µg/mL (0.6 µM) against FCV, which is within the range of values found when tested on other viruses. Recently, the drug was reported to inhibit GI norovirus replicon replication at 5 µg/mL, and cleared the replicon from the host cells, but was ineffective against murine norovirus [64].
Nitazoxanide has been commercialized in Latin American countries and India to treat a broad spectrum of intestinal parasitic infections and is currently in clinical trials to treat norovirus gastroenteritis [54,63]. For example, a large randomized, double-blind, placebo-controlled clinical trial is being conducted using nitazoxanide to treat acute gastroenteritis mainly caused by Cryptosporidium parvum, norovirus, and rotavirus in hospitalized aboriginal children in the Northern Territory, Australia [65]. There is also some published anecdotal evidence that this drug works on norovirus in a small number of case studies [66,67].
In the veterinary field, small animals such as cats and dogs have received nitazoxanide to treat intestinal parasites. Gookin et al. [68] demonstrated the successful use of nitazoxanide in eliminating the shedding of Tritrichomonas foetus, a cause of chronic diarrhea in cats. In another study, the successful administration of nitazoxanide to treat giardiasis and cryptosporidiosis in dogs was demonstrated [69]. Given that nitazoxanide displayed a potent inhibition against FCV and is already used in a clinical setting for feline infections, our data illustrate that nitazoxanide could be repurposed for the treatment of FCV infections. However, considering the narrow in vitro therapeutic index of nitazoxanide, and its side-effects (diarrhea and vomiting) observed in cats after nitazoxanide administration [68], concerns about the effective dose in vivo should be addressed.
The combination of antiviral compounds with additive or synergistic effects is a strategy to improve drug efficacy, reduce antiviral toxicity, and limit the development of viral resistance. Here, we demonstrated that the combination of nitazoxanide and 2CMC in cell cultures had a synergistic inhibitory effect against FCV, with an average delta score of 7.79 ( Figure 4D). As nitazoxanide showed cytotoxicity on CRFK cells at a relatively low concentration (CC 50 = 12.7 µM), the synergistic effect resulted from the combination with 2CMC (CC 50 > 100 µM) could be useful in limiting its cytotoxic effects by reducing the effective concentration of nitazoxanide, and overall improving the efficacy of the combination treatment.
In the present study, we have expressed the recombinant FCV Pro-Pol with high yields of active protein. Previous studies have demonstrated that the fusion protein is stably expressed in FCV-infected cells and is the primary and active form of the protein, which maintains both protease and polymerase activity [18,70]. As previously shown by Wei et al. [18], we also demonstrated that high concentrations of NaCl (100 mM) caused a reduction in the RdRp activity ( Figure 1B), however, no effect in the protease activity was observed at this concentration ( Figure 3B).
Of the six NNI compounds tested in the current study, PPNDS and quercetagetin showed an inhibition of FCV RdRp activity with IC 50 values in the low micromolar range (Figure 2 and Table 1). In previous studies, PPNDS demonstrated potent inhibition of RdRp activity against viruses from three calicivirus genera, Norovirus, Sapovirus, and Lagovirus, with IC 50 values between 0.1 and 2.3 µM [33,42,71]. However, due to cell permeability issues limiting bioavailability and antiviral efficacy in cell cultures, PPNDS is not considered a potential antiviral drug candidate [72,73]. While in the current study quercetagetin displayed an IC 50 of 2.8 µM in polymerase assays, it did not inhibit FCV plaque formation and therefore is not a suitable FCV antiviral. Quercetagetin, a natural flavonoid compound, was first reported as a potent inhibitor of HCV replication in vitro [74]. The compound demonstrated a potent RdRp inhibition against different HCV genotypes, with IC 50 s between 2.8 and 6.1 µM, but was less potent in cell cultures against the infectious virus (EC 50 40.2 µM ± 17.7) [74]. Quercetagetin also showed a moderate inhibitory activity against the chikungunya replicon, with an IC 50 of 43.5 µM [75].
In addition to the polymerase inhibition assay, using the purified FCV Pro-Pol, we also described a FRET protease assay for high throughput screening of FCV protease inhibitors. As with viral polymerases, proteases play a crucial role in the viral replication cycle and are attractive targets for antiviral development. Several viral PIs are currently approved or under development to treat pathogenic viruses such as HIV, HCV, and the SARS coronavirus [76,77]. GC376 is under development for feline coronavirus infection (feline infectious peritonitis) [78]. Using the FRET-based assay, we tested three previously published PIs, with only GC376 demonstrating a moderate inhibition against the protease (IC 50 of 18.7 µM) ( Figure 3C). This compound has previously shown a potent inhibition against the proteases of norovirus, coronaviruses, and picornaviruses, with IC 50 s ranging from 0.20 to 4.35 µM [29]. However, against FCV in cell-based assays, an EC 50 value of 35 µM was obtained [29], similar to the value obtained in our study. The PIs rupintrivir and chymostatin have previously demonstrated an inhibition of the human norovirus protease (genogroup I and II) in FRET-based assays, with IC 50 values of <1 µM and 5-10 µM, respectively [29,52]. However, no inhibitory effect was observed for either PI against FCV protease in this study.
Among the NAs tested, famciclovir is used for the treatment of feline herpesvirus (FHV)-associated clinical disease [79]. This drug is also commercially used as an FCV and FHV treatment. We tested famciclovir at concentrations up to 50 µM using the cell culture plaque reduction assay with no antiviral effect observed. Our data show that the compound is ineffective at inhibiting virus replication and thus is a poor therapeutic option for the treatment of FCV infections.
FCV is a highly infectious respiratory pathogen of cats with a global distribution, and more recently FCV-VSD associated high-mortality outbreaks have been reported. Despite the availability of a vaccine, the high diversity of the FCV genome plays a key role in vaccine failure and is also the basis for the emergence of virulent strains. In addition, there are currently no approved antivirals to treat the disease. Here, we report the establishment of two in vitro assays that allow for the identification of novel inhibitors of the FCV polymerase and protease. The present findings have implications for the development of FCV antivirals, providing a basis to design and select drugs which may be used in the veterinary clinic. Using the in vitro assays, we identified quercetagetin and PPNDS as potent RdRp inhibitors, and we also demonstrated a moderate inhibition of protease activity by GC376. Finally, we reported the identification of two compounds (nitazoxanide and 2CMC) with antiviral activity against FCV in cell culture at low micromolar concentrations with a potential combinational therapeutic utility to treat FCV-infected cats.