key: cord-0004031-24lz9tf1
authors: Zhou, Hongzhuan; Su, Xia; Lin, Lulu; Zhang, Jin; Qi, Qi; Guo, Fangfang; Xu, Fuzhou; Yang, Bing
title: Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells
date: 2019-08-13
journal: Viruses
DOI: 10.3390/v11080742
sha: 436164085d9fd52ea5d7ebed65465e0a41495c04
doc_id: 4031
cord_uid: 24lz9tf1

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs—Nitazoxanide, Closantel Sodium, and Closantel—with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

Canine parvovirus (CPV), genus Protoparvovirus, a member of the family Parvoviridae (subfamily Parvovirinae), is a small, highly contagious, nonenveloped, single-stranded DNA virus [1] . CPV is a major causative agent of acute gastroenteritis, leukopenia and myocarditis in dogs, and typical clinical signs include vomiting, fever, and diarrhea. Generally, puppies aged 6 weeks to 6 months have been found to be more susceptible to CPV infection [2, 3] .

The CPV genome is approximately 5.2 kb in length, and contains two open reading frames (ORF), which encode 2 nonstructural proteins (NS1 and NS2) and two structural proteins (VP1 and VP2) [4] [5] [6] .

indicating 100% CPE inhibition, and OD 450 values of wells with SD6 infection served as a negative (virus) control indicating 0% CPE inhibition [14] . The percentage inhibition was calculated using the formula: percentage CPE inhibition = (OD 450 of drug treated cells − OD 450 of negative control)/(OD 450 of positive control − OD 450 of negative control) × 100 [20, 21] . All drug plates were set-up in duplicates for primary screening, and drugs show greater than 20% CPE inhibition from the primary screen were used for second round of screening. The further validation of identified drugs was conducted in triplicates, and drugs show greater than 50% CPE inhibition were used for further analysis.

Dose response experiments were performed to test CC 50 s and EC 50 s of drugs as described above with minor modifications. For CC 50 assays, 96 µL of F81 cells (2.5 × 10 4 cells/well) was mixed with 4 µL prediluted drugs at final concentrations ranging from 0.0024-160 µM. For EC 50 assays, 86 µL of F81 cells (2.5 × 10 4 cells/well) was pretreated with 4 µL prediluted drugs at final concentrations ranging from 0.0024-160 µM for 1 h, and then treated cells were infected with 10 µL CPV at an MOI of 0.076. Both CC 50 and EC 50 assays were done in triplicate. Cell cytotoxicity and inhibition of CPV infection were both examined after 40 h incubation using the TransDetect ® Cell Counting Kit (TransGen Biotech, Beijing, China). The CC 50 and EC 50 values were calculated via a best-fit Log (dose)-response curve-fitting in GraphPad Prism software (version 7.00, La Jolla, CA, USA) [21] .

Absolute quantification PCR was used to evaluate the anti-CPV effect. Total DNA was isolated from SD6 infected F81 cells using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacture's instruction. CPV specific primers VP2-F 5 -CAAATAGAGCATTGGGCTTACC-3' and VP2-R 5'-TCCCATTTGAGTTACACCACG-3' were used to amplify the 119-bp fragment. The obtained fragment was cloned into pMD ® 18-T (Takara, Shiga, Japan), and the resulting positive clone was named pMD-VP2S for further use. To evaluate the antiviral effects of the three drugs of Nitazoxanide, Closantel Sodium and Closantel, F81 cells were seeded in 6-well plates at 7.5 × 10 5 cells per well and pretreated with the 3 drugs at a final concentrations of 5 µM, 10 µM, and 20 µM, respectively, for 1 h, then treated cells were infected with CPV at MOI of 0.076 as described above. Cells treated with 0.1% DMSO were used as control. After 40 h incubation, total DNA was extracted from whole cell lysates by QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The quantitative standard curve was generated via quantitative real-time PCR of the plasmid pMD-VP2S preparations at serial dilutions of 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 copies/µL. Each 20 µL qPCR reaction mixture contained 1 µL 10-fold diluted sample, 10 µL SuperReal PreMix Plus (SYBR Green) (TianGen Biotech, Beijing, China), and 0.2 µM of specific primers. All mixtures were then loaded into a StepOne Plus qPCR machine (Applied Biosystems, USA). The qPCR procedure was of 3 min at 95 • C, followed by 45 cycles of 5 s at 95 • C, and 30 s at 60 • C. The absolute number of DNA copies of the VP2 gene in the cells was determined according to the generated standard curve.

Immunofluorescence assay (IFA) was used to further evaluate the antiviral effects of identified drugs. F81 cells in 96-well plates (2.5 × 10 4 cells/well) were pretreated with 4 µL prediluted Nitazoxanide, Closantel Sodium, and Closantel at final concentrations of 5 µM, 10 µM, and 20 µM, respectively, for 1 h, then treated cells were infected with 10 µL CPV at MOI of 0.076. After 30 h postinfection, cells were fixed with 80% acetone, and then incubated with a 1:100 dilution of mouse anti-VP2 monoclonal antibody (INGENASA, Madrid, Spain) for 40 min, followed by incubation with a 1:200 dilution of fluorescein isothiocyanate-conjugated Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (Invitrogen, Carlsbad, CA, USA). Finally, the cells were stained with 4 ,6-diamidino-2-phenylindole (DAPI) in order to label cell nuclei in focus. After washing, the cells were examined with High Content imaging System (Operetta, PerkinElmer, Waltham, MA, USA) at 20× magnification.

The time of addition experiment was used to test the drug inhibition stage of the CPV replication life cycle. Meanwhile, the inhibitory effects of the identified drugs at different time points following the addition of the three drugs after virus infection were also evaluated by this assay. Briefly, F81 cells were seeded in 96-well plates (2.5 × 10 4 cells/well) and then infected with CPV (MOI = 0.076). Nitazoxanide, Closantel Sodium, Closantel, or 0.1% DMSO were added at −1 h (1 h pre-infection), 0 h (CPV infection), 0.5 h, 1 h, 2 h, 3 h or 6 h (post-infection) to determine the inhibitory effects at different time points of drug addition [20] [21] [22] . Cells treated with 0.1% DMSO served as control, and the effect on drug inhibition was evaluated using TransDetect ® Cell Counting Kit (TransGen Biotech, Beijing, China) at 40 h postinfection, as described above.

Western blot was also used to evaluate the antiviral activity of Nitazoxanide, Closantel Sodium, and Closantel against different subspecies of CPV variants. F81 cells were seeded in 6-well plates at 7.5 × 10 5 cells per well and pretreated with the three drugs at final concentrations of 5 µM, 10 µM, and 20 µM, respectively, for 1 h. The treated cells were infected with CPV variants SD6 (New CPV-2a strain), SD3 (New CPV-2b strain) and BJ-1 (New CPV-2a strain), at an MOI of 0.076 as described above. Cells with 0.1% DMSO were used as control. After 40 h incubation, cells were harvested and lysed with ProteinExt ® Mammalian Total Protein Extraction Kit (TransGen Biotech, China). Equal amounts of cell lysates were analyzed via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocking with 5% milk-TBS-Tween 20 for 1 h at room temperature, anti-VP2 monoclonal antibody (1:800 dilution, INGENASA, Madrid, Spain), and beta-actin monoclonal antibody (AC-15) (1:4000 dilution, Thermo Scientific, Waltham, MA, USA) were added and incubated overnight at 4 • C, blots were further incubated with horseradish-peroxidase(HRP)-conjugated goat anti-mouse IgG for 1 h at 37 • C. The immunoreactive bands were detected using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate Kit (Thermo Scientific, USA) and imaged using a chemiluminescence apparatus (Proteinsimple, USA). Band intensities were measured using the Image J software, and viral VP2 protein expression was first compared with beta Actin expression, and then normalized to the 0.1% DMSO-treated group.

F81 cells were seeded in 96-well plates (2.5 × 10 4 cells/well) and pretreated with 4 µL Nitazoxanide, Closantel Sodium, and Closantel with final concentrations of 10 µM, then infected with CPV (MOI = 0.076). Cells were incubated for 4 h, 8 h, 12 h and 24 h at 37 • C and 5% CO 2 . The assay was performed in triplicates. Caspase-Glo 3/7 assay kit (Promega, USA) was used to detect pro-or antiapoptotic effects of the identified drugs [14, 23] . The luminescence of each sample was measured using a SYNERGY H1 microplate reader (BioTek Instruments Inc., Winooski, USA) according to the manufacturer's instructions [14] .

The CC 50 s and EC 50 s of drugs were determined by a best-fit Log(dose)-response curve-fitting in GraphPad Prism 7. One-way analysis of variance (ANOVA) and Dunnett's multiple comparisons test were used to analyze data. Statistical significances are denoted as follows; * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001. 

In this study, a CPE-based high-throughput screening assay was used to screen CPV inhibitors from an FDA-approved drug library. The timeline of drug treatment and CPV infection, as well as the flow chart of the CPE-based assay, are shown in Figure 1A ,B. In the primary screen (First round), the Z' factor was between 0.68 and 0.83 across all 17 drug plates. As the assay quality control index Z' factors were >0.5 in all plates, it demonstrated that the CPE-based screening assay was suitable for screening anti-CPV drugs. The mean percentage CPE inhibition of each drug was plotted in Figure 1C .

Viruses 2019, 11, x FOR PEER REVIEW 5 of 14 round), the Z' factor was between 0.68 and 0.83 across all 17 drug plates. As the assay quality control index Z' factors were >0.5 in all plates, it demonstrated that the CPE-based screening assay was suitable for screening anti-CPV drugs. The mean percentage CPE inhibition of each drug was plotted in Figure 1C . Twenty-one drugs showing >20% CPE inhibition from the primary screen were used for a second round of screening, and seven drugs with percentage inhibition >50% were further identified. (C) Scatter plot of percentage CPE inhibition results for 1430 FDA-approved drugs, numbers in X axis mean the species of the tested drugs, each number corresponds to a specific drug, and the order is the same as that provided in the manual of the FDA-approved drug library, each dot shows the mean percentage CPE inhibition in the presence of 10 μM tested drug.

Twenty-one drugs with >20% CPE inhibitions, identified during the first round of screening, were used for the second round of screening. The drug name, catalogue number of Selleck, and the final percentage CPE inhibition of the 21 drugs are listed in Table S1 , and the inhibitory effects of these drugs, when these drugs were added 1 h post-virus infection are also listed in Table S1 . Seven drugs with percentage CPE inhibitions >50% were selected for further CC50 and EC50 assays, and the results are shown in Figures 2 and S1 and are also listed in Table 1 . Table 1 . 50% cytotoxicity concentration (CC50), 50% antiviral efficacy concentration (EC50), and selectivity index (SI) of identified anti-CPV drugs. Flow chart of drug screen using CPE-based assay. Briefly, F81 cells per well were pretreated with 10 µM drugs for 1 h, and then infected with 0.076 MOI CPV, cell viability was detected at 40 h postinfection as described above, antiviral inhibitors against CPV were determined according to the percentage CPE inhibition. Twenty-one drugs showing >20% CPE inhibition from the primary screen were used for a second round of screening, and seven drugs with percentage inhibition >50% were further identified. (C) Scatter plot of percentage CPE inhibition results for 1430 FDA-approved drugs, numbers in X axis mean the species of the tested drugs, each number corresponds to a specific drug, and the order is the same as that provided in the manual of the FDA-approved drug library, each dot shows the mean percentage CPE inhibition in the presence of 10 µM tested drug.

Twenty-one drugs with >20% CPE inhibitions, identified during the first round of screening, were used for the second round of screening. The drug name, catalogue number of Selleck, and the final percentage CPE inhibition of the 21 drugs are listed in Table S1 , and the inhibitory effects of these drugs, when these drugs were added 1 h post-virus infection are also listed in Table S1 . Seven drugs with percentage CPE inhibitions >50% were selected for further CC 50 and EC 50 assays, and the results are shown in Figure 2 and Figure S1 and are also listed in Table 1 . The top three drugs-Nitazoxanide, Closantel Sodium, and Closantel-were identified with higher percentage CPE inhibition of 106.59 ± 2.79, 69.76 ± 6.06, and 80.64 ± 7.87%, respectively, at 10 μM concentration (Table S1 ). Moreover, all three drugs showed a dose-dependent inhibition of CPV infection ( Figure 2 ). 

From the absolute qPCR results, dose-dependent reduction in the copy numbers of CPV viral DNA were observed with increasing concentrations of Nitazoxanide ( Figure 3A ), Closantel Sodium, ( Figure 3B ) or Closantel ( Figure 3C ). When CPV-infected F81 cells were treated with the three drugs at 10 μM, the CPV viral DNA copy numbers of 1mL whole cell lysates significantly reduced to 0.07% (Nitazoxanide), 24.04% (Closantel Sodium), and 20.83% (Closantel) compared with the 0.1% DMSO-treated group ( Figure 3) . The top three drugs-Nitazoxanide, Closantel Sodium, and Closantel-were identified with higher percentage CPE inhibition of 106.59 ± 2.79, 69.76 ± 6.06, and 80.64 ± 7.87%, respectively, at 10 µM concentration (Table S1 ). Moreover, all three drugs showed a dose-dependent inhibition of CPV infection ( Figure 2 ).

From the absolute qPCR results, dose-dependent reduction in the copy numbers of CPV viral DNA were observed with increasing concentrations of Nitazoxanide ( Figure 3A ), Closantel Sodium, ( Figure 3B ) or Closantel ( Figure 3C ). When CPV-infected F81 cells were treated with the three drugs at 10 µM, the CPV viral DNA copy numbers of 1mL whole cell lysates significantly reduced to 0.07% (Nitazoxanide), 24.04% (Closantel Sodium), and 20.83% (Closantel) compared with the 0.1% DMSO-treated group ( Figure 3) .

As shown in Figure 4 , CPV infection could be inhibited in the presence of Nitazoxanide, Closantel Sodium, and Closantel at a concentration of 5 µM. Few cells were CPV-positive when treated with 10 µM of the drugs. Almost no green signals were detected in all F81 cells treated with 20 µM of the drugs. The results confirmed that these identified drugs inhibited CPV infection in a dose-dependent manner, which was consistent with the qPCR assay results.

From the absolute qPCR results, dose-dependent reduction in the copy numbers of CPV viral DNA were observed with increasing concentrations of Nitazoxanide ( Figure 3A ), Closantel Sodium, ( Figure 3B ) or Closantel ( Figure 3C ). When CPV-infected F81 cells were treated with the three drugs at 10 μM, the CPV viral DNA copy numbers of 1mL whole cell lysates significantly reduced to 0.07% (Nitazoxanide), 24.04% (Closantel Sodium), and 20.83% (Closantel) compared with the 0.1% DMSO-treated group (Figure 3) . As shown in Figure 4 , CPV infection could be inhibited in the presence of Nitazoxanide, Closantel Sodium, and Closantel at a concentration of 5 μM. Few cells were CPV-positive when treated with 10 μM of the drugs. Almost no green signals were detected in all F81 cells treated with 20 μM of the drugs. The results confirmed that these identified drugs inhibited CPV infection in a dose-dependent manner, which was consistent with the qPCR assay results. 

Consistent with screening results, all three drugs showed anti-CPV effects when added 1 h before virus infection (pre-infection). The inhibitory effects of Nitazoxanide, Closantel Sodium, and Closantel were 108.00 ± 17.74, 58.14 ± 7.28, and 71.28 ± 3.17, respectively, when the three drugs were added 1 h post-virus infection ( Figure 5 ). The drugs inhibited the early processes of the CPV 

Consistent with screening results, all three drugs showed anti-CPV effects when added 1 h before virus infection (pre-infection). The inhibitory effects of Nitazoxanide, Closantel Sodium, and Closantel were 108.00 ± 17.74, 58.14 ± 7.28, and 71.28 ± 3.17, respectively, when the three drugs were added 1 h post-virus infection ( Figure 5 ). The drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection ( Figure 5 ). In addition, the anti-CPV activity of Nitazoxanide was observed when the drug was added 3 h postinfection, suggesting that Nitazoxanide may partially have the ability to inhibit CPV infection at the stage of viral replication. 

Western blot was also used to evaluate the broad-spectrum antiviral activity of identified drugs against different subspecies of three CPV variants. Dose-dependent reductions in VP2 expression and the quantification of relative expression levels are shown in F81 cells treated with Nitazoxanide ( Figure 6A,D) , Closantel Sodium ( Figure 6B ,E) or Closantel ( Figure 6C,F) , respectively. Nitazoxanide treated at 10 μM reduced relative expression of VP2 in different CPV variants to 9.68% (SD6), 36.29% (SD3), and 11.22% (BJ-1), respectively ( Figure 6D ). Closantel Sodium treated at 10 μM reduced relative expression of VP2 in different CPV variants to 22.50% (SD6), 23.85% (SD3) and 12.83% (BJ-1), respectively ( Figure 6E ). Closantel treated at 10 μM reduced relative expression of VP2 in different CPV variants to 30.1% (SD6), 10.78% (SD3), and 14.58% (BJ-1), respectively ( Figure 6F ). All the identified drugs showed inhibitory ability against CPV variants SD6, SD3, and BJ-1. 

Western blot was also used to evaluate the broad-spectrum antiviral activity of identified drugs against different subspecies of three CPV variants. Dose-dependent reductions in VP2 expression and the quantification of relative expression levels are shown in F81 cells treated with Nitazoxanide ( Figure 6A,D) , Closantel Sodium ( Figure 6B ,E) or Closantel ( Figure 6C,F) , respectively. Nitazoxanide treated at 10 µM reduced relative expression of VP2 in different CPV variants to 9.68% (SD6), 36.29% (SD3), and 11.22% (BJ-1), respectively ( Figure 6D ). Closantel Sodium treated at 10 µM reduced relative expression of VP2 in different CPV variants to 22.50% (SD6), 23.85% (SD3) and 12.83% (BJ-1), respectively ( Figure 6E ). Closantel treated at 10 µM reduced relative expression of VP2 in different CPV variants to 30.1% (SD6), 10.78% (SD3), and 14.58% (BJ-1), respectively ( Figure 6F ). All the identified drugs showed inhibitory ability against CPV variants SD6, SD3, and BJ-1. 

Doley et al. (2014) reported that CPV could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in Madin-Darby canine kidney (MDCK) cells [24] . In order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase-3 activity of drug-treated cells with or without CPV infection. As shown in Figure 7 , the antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells with or without CPV infection. Therefore, Nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. Closantel Sodium-or Closantel-treated cells had no pro-or antiapoptotic effects ( Figure S2) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. Statistical analysis was carried out using one-way ANOVA and Dunnett's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001 (compared to 0.1% DMSO-treated cells without CPV Figure 6 . Potential broad-spectrum anti-CPV activity of identified drugs. F81 cells were seeded in 6-well plates and pretreated with 5 µM, 10 µM, or 20 µM of Nitazoxanide (A), Closantel Sodium (B), or Closantel (C) for 1 h, respectively. Then treated cells were infected with various CPV strains SD6, SD3 or BJ-1. Cells were harvested and lysed at 40 hpi for western blot analysis. Band intensities were then measured using software Image J, and VP2 expression was analyzed and compared to beta Actin expression. Relative expression levels for Nitazoxanide-(D), Closantel Sodium-(E), and Closantel (F)-treated results are presented in bar graphs. Error bars represent standard errors from three independent experiments. Statistical analysis was compared to Control (0.1% DMSO-treated cells, marked as D in the figure) and carried out using one-way ANOVA and Dunnett's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Doley et al. (2014) reported that CPV could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in Madin-Darby canine kidney (MDCK) cells [24] . In order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase-3 activity of drug-treated cells with or without CPV infection. As shown in Figure 7 , the antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells with or without CPV infection. Therefore, Nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. Closantel Sodium-or Closantel-treated cells had no pro-or antiapoptotic effects ( Figure S2) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. 

Doley et al. (2014) reported that CPV could induce caspase-dependent (involving extrinsic, intrinsic, and endoplasmic reticulum pathways) apoptosis in Madin-Darby canine kidney (MDCK) cells [24] . In order to evaluate whether drug-associated apoptosis was involved in the antiviral effect, we measured the caspase-3 activity of drug-treated cells with or without CPV infection. As shown in Figure 7 , the antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells with or without CPV infection. Therefore, Nitazoxanide-associated caspase activation reduction (apoptosis) might be involved in the antiviral effect of the drug. Closantel Sodium-or Closantel-treated cells had no pro-or antiapoptotic effects ( Figure S2) ; hence, the antiviral effects of these two drugs may not be due to drug-associated apoptosis. 

CPV is a widely distributed virus and contains at least three main subspecies: CPV-2a, CPV-2b, and CPV-2c [4, 6, 8] . Currently, commercial vaccines cannot provide complete protection against all CPV variants. Moreover, no effective drug is available to control CPV infection except for supportive and symptom-based care. Hence, it is important to develop an alternative treatment against CPV infection. In this study, we developed a CPE-based assay to screen CPV inhibitors from a FDA-approved drug library, and successfully identified three FDA-approved CPV inhibitors. These drugs might provide potential treatment options for anti-CPV infections.

Although the selectivity index (SI) of Gemcitabine HCl, Cladribine, Gemcitabine, and Trifluridine were at a higher level (Table 1) , the percentage CPE inhibition of the four drugs was always maintained at a lower level ( Figure S1 ). The maximum percentage CPE inhibitions for the drugs were between 51.80 ± 2.48 and 68.37 ± 7.79, which are relatively lower CPE inhibition levels that would not increase with increased drug concentration. Therefore, Nitazoxanide, Closantel Sodium, and Closantel were selected for further study.

As mentioned above, the identified drugs Nitazoxanide, Closantel Sodium, and Closantel can reduce the copy numbers of CPV viral DNA to 0.07%, 24.04%, and 20.83%, respectively, compared with the 0.1% DMSO-treated control ( Figure 3) . Meanwhile, the IFA result also showed that these identified drugs inhibited CPV infection in a dose-dependent reduction manner. Western blot showed that 10 µM Nitazoxanide treatment reduced relative VP2 expression in three CPV variants SD6, SD3, and BJ-1 to 9.68%-36.29%, and the reduction rates following 10 µM Closantel Sodium and Closantel treatment were 12.83%-23.85% and 10.78%-30.1%, respectively ( Figure 6 ). These results indicated that the identified drugs had significant inhibitory effects against CPV infection in F81 cells.

In previous studies, two drugs Oseltamivir and Cidofovir were added in the second round of screening. As a neuraminidase (NA) inhibitor, Oseltamivir has been used to treat the human influenza virus. Savigny and Macintire (2010) used Oseltamivir for CPV enteritis and found that the Oseltamivir-treated group gained a significant increase of weight and had no changes in white blood cell (WBC) count compared to the control group; however, the authors also reported that no obvious advantage had been established [25] . Cidofovir is a broad-spectrum anti-DNA virus drug, which had been evaluated for the treatment of human papillomavirus (HPV)-associated tumors [26] . Our CPE-based screening assay showed that the percentage CPE inhibition of Oseltamivir and Cidofovir were 2.13 ± 2.41 and −1.28 ± 1.03 (Table S1 ), respectively, and that these two drugs had no anti-CPV effects on F81 cells.

Previously, Nitazoxanide was used to treat cryptosporidiosis, giardiasis, and other parasitic infections [27] . Recently Nitazoxanide was reported to inhibit various DNA and RNA viruses, including hepatitis B virus (HBV) [28, 29] , human cytomegalovirus (HCMV) [30] , influenza A virus [31] , hepatitis C virus [32] , norovirus [33] , rotavirus [34] , Japanese encephalitis virus (JEV) [35] , coronavirus [36] chikungunya virus (CHIKV) [20] , human immunodeficiency virus (HIV) [37] , and ZIKV [38] .

The antiviral mechanism of Nitazoxanide remains unclear for now. Nitazoxanide could impair the terminal glycosylation of the influenza A hemagglutinin protein or the formation of E1-E2 (Rubella virus surface glycoproteins) complex of the Rubella virus (RV), thus affecting the assembly of influenza A virus and RV, respectively [31, 39] . In addition, Nitazoxanide could also hinder the interactions between the proteins NSP5 and NSP2 of Rotavirus or the interactions between proteins NS2B and NS3 of ZIKV and dengue virus 2 (DENV2) [34, 40] . Mercorelli et al. (2016) also reported that Nitazoxanide can inhibit the transcriptional activation properties of the HCMV immediate-early 2 (IE2) protein [30] . These results indicated the virus-specific effects of Nitazoxanide.

Since Nitazoxanide can inhibit the replication of various DNA and RNA viruses, various studies have focused on identifying host factors to explain the broad antiviral activities of Nitazoxanide. Ashiru et al. (2014) reported that Nitazoxanide depleted intracellular Ca 2+ stores, besides the phosphorylation of PKR and eIF2α, further affecting N-linked glycosylation of the bovine viral diarrhea virus (BVDV) E2 protein and trafficking from the ER to the Golgi [41] . Nitazoxanide can elicit antiviral innate immunity and reduce the HIV replication by activating the interferon system and further expression of various interferon-stimulated genes (ISGs) [37] . In addition, Nitazoxanide might block the production of acetyl-CoA, which is a required metabolic intermediates for Vaccinia virus (VACV) reproduction. In general, further studies are still required to clearly elucidate the antiviral mechanism of Nitazoxanide [27] .

Closantel sodium and Closantel were also identified and shown to have anti-CPV activities. Closantel is a salicylanilide derivative and is considered to be an anthelmintic agent in livestock [42] . The antiangiogenesis and anticancer effects of Closantel sodium and Closantel have also been previously reported [43] . However, to our knowledge, neither the antiviral activity nor the antiviral mechanisms of Closantel sodium and Closantel have been reported before. Previous studies have shown that Closantel inhibited B-Raf (a serine/threonine kinase) V600E [44] , adenine nucleotide translocase (ANT) [45] , SPAK and OSR1 kinase [46] . In addition, Senkowski et al. (2015) reported that Closantel could inhibit mitochondrial respiration as well [47] . These reports may contribute to further studies on the antiviral mechanism of Closantel.

In this study, which is aimed at identifying anti-CPV drugs for potential therapeutic use, a CPE-based assay was developed for screening CPV inhibitors from an FDA-approved drug library. After screening, the top three drugs, Nitazoxanide, Closantel Sodium, and Closantel, with higher percentage CPE inhibition, were selected and further confirmed by qPCR and IFA. In addition, the identified drugs can inhibit different subspecies of CPV variants and displayed broad-spectrum antiviral activity against CPV. Hence, these drugs may provide potential options for the treatment of CPV infection.

The following are available online at http://www.mdpi.com/1999-4915/11/8/742/s1, Table S1 : Percentage inhibition (%) of 21 drugs and 2 other tested drugs, Figure S1 : Evaluation of cytotoxicity and anti-CPV efficacy of other four drugs, Figure S2 

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Identification of FDA-approved drugs that target hepatitis B virus transcription

Antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission

Identification of Three Antiviral Inhibitors against Japanese Encephalitis Virus from Library of Pharmacologically Active Compounds 1280

Identification of pyrrolo[3,2-c]pyridin-4-amine compounds as a new class of entry inhibitors against influenza viruses in vitro

Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity

Canine parvovirus type 2a (CPV-2a)-induced apoptosis in MDCK involves both extrinsic and intrinsic pathways

Use of oseltamivir in the treatment of canine parvoviral enteritis

The broad-spectrum anti-DNA virus agent cidofovir inhibits lung metastasis of virus-independent, FGF2-driven tumors

Inhibition of vaccinia virus replication by nitazoxanide

Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication

Thiazolides as Novel Antiviral Agents. 1. Inhibition of Hepatitis B Virus Replication

Drug Repurposing Approach Identifies Inhibitors of the Prototypic Viral Transcription Factor IE2 that Block Human Cytomegalovirus Replication

Thiazolides, a New Class of Anti-influenza Molecules Targeting Viral Hemagglutinin at the Post-translational Level

Treatment of chronic viral hepatitis with nitazoxanide and second generation thiazolides

Norovirus Gastroenteritis Successfully Treated with Nitazoxanide

Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation

Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model

A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs

Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

Pediatric Drug Nitazoxanide: A Potential Choice for Control of Zika

Inhibition of rubella virus replication by the broad-spectrum drug nitazoxanide in cell culture and in a patient with a primary immune deficiency

Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

Nitazoxanide, an antiviral thiazolide, depletes ATP-sensitive intracellular Ca(2+) stores. Virology

Discovery of novel SPAK inhibitors that block WNK kinase signaling to cation chloride transporters

Closantel Suppresses Angiogenesis and Cancer Growth in Zebrafish Models

Repositioning organohalogen drugs: A case study for identification of potent B-Raf V600E inhibitors via docking and bioassay

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

Rafoxanide and Closantel Inhibit SPAK and OSR1 Kinases by Binding to a Highly Conserved Allosteric Site on Their C-terminal Domains

Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer

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The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.