Accepted Manuscript

In vitro antiviral efficacy of caffeic acid against canine distemper virus

Zong-Mei Wu, Zhen-Jiang Yu, Zhen-Qiang Cui, Lu-Yuan Peng, Hao-Ran Li, Chun-Lei
Zhang, Hai-Qing Shen, Peng-Fei Yi, Ben-Dong Fu

PII: S0882-4010(17)30527-2
DOI: 10.1016/j.micpath.2017.07.006
Reference: YMPAT 2344

To appear in: Microbial Pathogenesis

Received Date: 10 May 2017

Revised Date: 29 June 2017
Accepted Date: 3 July 2017

Please cite this article as: Wu Z-M, Yu Z-J, Cui Z-Q, Peng L-Y, Li H-R, Zhang C-L, Shen H-Q, Yi P-F,
Fu B-D, In vitro antiviral efficacy of caffeic acid against canine distemper virus, Microbial Pathogenesis
(2017), doi: 10.1016/j.micpath.2017.07.006.

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IC50(µg/ml) SI
Compounds CC50(µg/ml)

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-1 h 0h 1h 2h -1 h 0h 1h 2h

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Caffeic acid >200 — — 23.3±0.9 32.3±0.7 — — >8.6 >6.2

RBV >200 — — 12.2±0.4 16.1±1 — — >16.4 >12.4

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1 In vitro antiviral efficacy of caffeic acid against canine
2 distemper virus
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4 Zong-Mei Wu#, Zhen-Jiang Yu#, Zhen-Qiang Cui, Lu-Yuan Peng, Hao-Ran Li,
5 Chun-Lei Zhang, Hai-Qing Shen, Peng-Fei Yi*, Ben-Dong Fu
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7 Department of Clinical Veterinary Medicine, College of Veterinary Medicine,

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8 Jilin University, No. 5333 Xi'an Road, Changchun, Jilin 130062, China

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# Zong-Mei Wu and Zhen-Jiang Yu contribute equally to this paper.

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12 * Corresponding author: Peng-Fei Yi

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13 Department of Clinical Veterinary Medicine
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14 College of Veterinary Medicine

15 Jilin University
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16 No. 5333 Xi'an Road, Changchun,

17 Jilin 130062, China

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18 Tel: +86-431-87835379
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19 E-mail address: yipengfei1982@126.com

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31 Abstract

32 Canine distemper (CD) is a highly contagious disease caused by the canine

33 distemper virus (CDV), and mortality can be as high as 100%. However, there is no

34 specific treatment for CD. In this study, the antiviral activity of the caffeic acid against

35 CDV was evaluated in vitro. The results showed that the IC50 of the caffeic acid

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36 against CDV at 1 and 2 h post infection (PI) is 23.3 and 32.3 µg/mL, respectively.

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37 Consistently, at 1 and 2 h PI, the caffeic acid exhibited a reduced (23.3-57.0% and

38 37.2-38.1%) viral inhibitory effect in vero cells. Furthermore, the caffeic acid plus

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39 Ribavirin (RBV) has greater antiviral activity against CDV than the caffeic acid or

40 RBV individually. In addition, the caffeic acid reduced the total viral RNA synthesis

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by 59-86% at 24-72 h. Therefore, our data provided the experimental evidence that
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42 the caffeic acid effectively inhibited CDV infection in vero cells, which may
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43 potentially be used to treat clinical disease associated with CDV infection.

44 Key words: canine distemper virus, caffeic acid, antiviral

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56 1. Introduction

57 Canine distemper virus (CDV) is a single-stranded RNA belonging to the genus

58 Morbillivirus family Paramyxoviridae. The incidence of canine distemper (CD) in

59 canine population seems to have increased in the past decades worldwide and even

60 though the disease is controlled by vaccination. However, many vaccinated dogs

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61 became distemper [1-3]. CDV causes systemic infections similar to but distinct from

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62 human measles in carnivores such as canines, felids, ferrets, raccoons and seals, with

63 lethality rates, depending on the host, of up to 100% [4, 5]. However, there is no

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64 specific antiviral agent for the treatment of CD. The nucleoside analogue 1-(b-D-

65 ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin, RBV) is the only

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commercially available molecule with a well-known antiviral activity towards several
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67 members of the Paramyxoviridae family [6-8]. Therefore, it is necessary to explore
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68 new agent to treat CD. The antiviral activity of the caffeic acid (3, 4-

69 dihydroxycinnamic acid) has been reported for some viruses, including influenza A
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70 virus [9]. Caffeic acid is an important phenolic compound commonly found in plants,
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71 foods, and propolis samples, particularly in the form of caffeic acid phenethyl ester

72 [10]. It is better known for its pharmacological properties, including antimicrobial,

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73 antioxidant, anti-inflammatory, and anticancer [11, 12].

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74 We hypothesized that the caffeic acid could have inhibitory effect on CDV.
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75 Therefore, in this study, our aim was to investigate the anti-CDV activity of the

76 caffeic acid in vitro.

77 2. Materials and methods

78 2.1. Cells and viruses

79 Vero cells were used for the in vitro growth of CDV. Vero cells were cultured in

80 DMEM with 5% fetal bovine serum (FBS) (Gibco) and antibiotics (100 units /ml

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81 penicillin, and 100 µg/ml streptomycin (Gibco, USA) at 37 °C and 5 % CO2

82 atmosphere incubator.

83 The CDV-11 strain (purchased from Qilu Animal Health Products Co., Ltd.) was

84 propagated in vero cells using a 5% FBS medium and subsequently titrated. The viral

85 titer was expressed as the 50% infectious dose of the tissue culture (TCID50/mL).

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86 Assessment of CDV growth in vero cells was performed after incubation at 37 °C and

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87 5 % CO2 for 4d.

88 2.2. Reagents

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89 Caffeic acid (Purity>98%) and RBV (Purity>98%) were purchased from the

90 National Institute for the Control of Pharmaceutical and Biological Products (Beijing,

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China), and their chemical structure were shown in Fig.1. They were dissolved in
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92 solution of dimethyl sulfoxide (DMSO) (Sigma, USA). The real-time RT-quantitative

PCR (qRT-PCR) and One Step PrimeScriptTM RT-PCR Kit were bought from Takara,
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94 Japan. All other chemicals were of reagent grade.

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95 2.3. Cytotoxicity assay

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96 The cytotoxicity of the tested compounds were performed on cells using a

97 colorimetric assay based on the MTT(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl)

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98 ( Amresco ) [13]. Vero cells were cultured into 96-well plate (106cells/well) of
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99 incubation at 37℃ and 5% CO2. The cells were treated with the tested compounds
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100 diluted in DMEM at different concentrations (from 12.5 to 200 µg/ml), after 72h

101 washed twice with phosphate-buffered saline (PBS), and incubated with 20µl of MTT

102 (5mg/mL) for 4h, the salt formed was solubilized by adding DMSO (150 mL/well)

103 and shaking for 10min. The optical density (OD) was determined using an EnSpire

104 Multimode plate Reader (PE, USA) with a 490-nm excitation filter. The 50%

105 cytotoxic concentration (CC50) values were calculated at least three independent

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106 experiments.

107 2.4. Antiviral assay

108 The intracellular activity of caffeic acid against CDV was evaluated with a

109 cytopathic effect (CPE) reduction assay on confluent vero cells, using the method of

110 Reed and Muench. Vero cells seeded in 96-well plates were infected with CDV at

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111 37 ℃ in 5 % CO2, and the tested compounds (12.5 to 200 µg/ml) were added at

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112 different time post infection (-1, 0, 1 and 2 h), according to the study [14]. The 50%

113 inhibitory concentration (IC50) was defined as the compound concentration required to

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114 reduce viral CPE by 50% of the virus control. The IC50 values of caffeic acid and

115 RBV were calculated as the mean±SD at least three independent experiments. The

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selectivity index (SI) was obtained by calculating the ratio of the CC50/IC50.
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117 2.5. Time-of-addition study

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118 To investigate the antiviral effect of caffeic acid at different stage, 96-well

119 microplates were seeded with vero cells monolayers (106 cells/well), incubated with
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120 the tested compounds at different time post infection (-1, 0, 1 and 2 h), vero cells were
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121 infected with dilutions of CDV (10-105 TCID50/mL). The study was according to the

122 method described in a previous study [14]. At the presence of 100 % CPE in the virus
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123 controls (approximately 72h PI), the antiviral effect was calculated using the method
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124 of Reed and Muench. The inhibition of CDV growth by the tested compounds was
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125 expressed as the TCID50 value at each time point. Assays were done in triplicate.

126 2.6. Real-time RT-quantitative PCR analysis

127 The reduction of viral growth in the presence of the tested compounds was evalu

128 ated using RNA quantification. Vero cells were seeded in 6-well plates were infected

129 with 500 TCID50 after an incubation time of 2h at 37 ℃ in 5% CO2, and vero cells we

130 re washed twice with PBS, then the tested compounds were added. After further incub

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131 ation of 24, 48 of 72h, the cells were harvested. One step Real Time PCR technique w

132 as used to quantify the copies of viral nucleic acid in the cells. The nucleotide sequenc

133 es of the forward and reverse primers were 5’-TGGTCGGAGAATTTAGAATGAAC

134 A-3’ and 5’-CACAAATCATTTCAGCAATTCTAGG-3’, respectively, and the TaqMa

135 n probe was CCAAGATGAGTGCCACCATGAACCGCC. The reaction included a fir

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136 st denaturation step at 42 ℃ for 5 min, 95 ℃ for 10s, followed by 40 cycles at 95 ℃ f

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137 or 5s, and 60 ℃ for 34s. Each sample and each dilution of the internal control were re

138 peated in duplicate during the same reaction, and the relative level of RNA expression

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139 was calculated as the mean of the two measurements.

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141 All values are expressed as means ± SD. Differences between mean values of

142 normally distributed data were analyzed using one-way ANOVA (Dunnett’s t-test).
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143 Statistical significance was accepted at P<0.05 or P<0.01.

144 3. Results
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145 3.1. The cytotoxicity of caffeic acid, RBV and caffeic acid plus RBV
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146 As shown in Fig. 2 A and B, the cytotoxicity was not observed in the cells

following the caffeic acid and RBV treatment for 72 h from 12.5 to 200 µg/ml
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148 compared with non-treated (control) cell. The CC50 values of the caffeic acid and
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149 RBV were >200 µg/ml (Table 1). There is also no cytotoxicity of the caffeic acid (32
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150 µg/ml) plus RBV (8-16 µg/ml) and DMSO (4%) (Fig. 2C).

151 3.2. Caffeic acid inhibits CDV infection in vero cells

152 When post-infection for 1 and 2 h, the caffeic acid showed antiviral effect. When

153 pre-infection for -1 and 0 h, no antiviral activity against CDV was observed (see Table

154 1).

155 The caffeic acid reduced viral yield at times 1h (IC50 23.3 µg/mL and SI >8.6),

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156 and 2h (IC50 32.3 µg/mL and SI >6.2) PI, and the efficiency of antiviral activity of

157 RBV was demonstrated at times 1 h (IC50 12.2 µg/mL and SI >16.4), and 2h (IC50

158 16.1 µg/mL and SI >12.4) PI.

159 3.3. The intracellular and extracellular antiviral activity of caffeic acid against

160 CDV

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161 To investigate the antiviral activity of caffeic acid against CDV, a time of

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162 addition assay was carried out by adding the caffeic acid at -1, 0, 1, and 2 h PI

163 (Fig.3). As shown in Fig. 3A, among the indicated concentrations, the caffeic acid

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164 did not have antiviral efficacy against CDV at -1 and 0 h PI. The antiviral effect of

165 the caffeic acid was demonstrated at 1 and 2 h PI. At 1 and 2 h PI, the caffeic acid

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(32 µg/mL) exhibited a reduced viral titer (23.4± 1.6% and 37.2 ± 5.6%) in vero cells.
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167 The 2-fold dose of the caffeic acid exhibited a reduced viral titer (57.0 ± 5.5% and
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168 38.1± 4.8%) at 1 and 2 h PI. As shown in Fig. 3B, RBV (8-16 µg/mL) did not have

169 antiviral efficacy against CDV at -1 h PI, while 32 µg/mL of RBV reduced 32.8 ±
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170 2.4% of TCID50. At 0 h PI, RBV (16-32 µg/mL) exhibited a reduced viral titer (14.2
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171 ± 0.1% and 23.1 ± 5.5%). At 1 and 2 h PI, RBV (16-32 µg/mL) exhibited a reduced

172 viral titer (32.98-100%). The caffeic acid displayed antiviral effects against CDV in a
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173 dose-dependent manner at 1h PI, and no significant differences at 2 h PI. The RBV
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174 displayed antiviral effects against CDV in a dose-dependent manner. In addition, we

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175 examined the antiviral activity of the caffeic acid (32 µg/mL) plus RBV (8-16 µg/mL)

176 against CDV infection in vero cells. At -1 h, the viral titer reduced from 36.8 ± 0.8%

177 to 59.4 ± 3.1%; at 0 h, the viral titer reduced from 14.3 ± 0.2% to 25.9 ± 2.7%; at 1 h,

178 the viral titer reduced from 12 ± 4% to 31.7 ± 1.7%, and at 2 h, the viral titer reduced

179 from 53.6 ± 3.6% to 52.7 ± 4.4%. The caffeic acid plus RBV has greater antiviral

180 activity against CDV than the caffeic acid or RBV individually at -1 h and 0 h PI, no

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181 significant differences at 1 h and 2 h PI (Fig. 3C).

182 3.4. Caffeic acid inhibits viral RNA accumulation during CDV replication cycle

183 Vero cells were infected with CDV (500 TCID50) and incubated for 24, 48 and

184 72h. The reduction of the total viral RNA was expressed as RNA copies/µL (Fig.4).

185 The results showed that the caffeic acid (32-64µg/mL) reduced the total viral RNA

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186 by 60.05– 61.48 %, 65.4-59.35 %, 66.58-86.99 % at 24, 48 and 72 h, respectively.

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187 4. Discussion

188 In this study, we investigated the antiviral activity of the caffeic acid against

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189 CDV and its possible machanisms of action.

190 Since RBV has been reported to have effect of high inhibiting CDV [6], it was of

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interest to assess whether the caffeic acid or the caffeic acid with RBV could also
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192 inhibit CDV. Our results showed that the caffeic acid in vitro had antiviral activity
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193 against CDV. Evaluation of viral growth depended on the observation of CPE, we

194 found that the IC50 of the caffeic acid against CDV at 1 and 2 h is 23.3 and 32.4 µg/ml,
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195 respectively compared with that of RBV as 12 and 16 µg/ml. The time of addition
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196 assay confirmed the antiviral activity of the caffeic acid is in a dose-dependent manner.

197 Consistent with this result, the caffeic acid aslo reduced viral RNA copies at different
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198 times PI. RBV is an example of such extrapolation and has demonstrated great
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199 antiviral efficiency against MeV[15]. Consistent with these findings, we found that
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200 RBV exhibited the inhibitory effect on CDV at lower concentrations (16-32 µg/ml).

201 Interestingly, except that the caffeic acid plus RBV inhibited the viral intracellular

202 stage of replicative cycle, the caffeic acid plus RBV also inhibited the extracellular

203 phase of the replicative cycle of CDV compared with the caffeic acid or RBV

204 individually. At -1 h PI, the RBV (8µg/ml) had no inhibitory effect on CDV, but the

205 caffeic acid plus RBV had works.

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206 The existence of different viral targets has been documented for RBV, such as

207 RNA polymerization and RNA capping enzymes for dengue virus, vaccinia virus and

208 hepatitis C virus [16-18]. We observed that the caffeic acid effectively inhibited the

209 multiplication of CDV. Coffee consumption is combined with a response to

210 peginterferon and ribavirin as a way for the treatment of hepatitis C [19]. The caffeic

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211 acid has been known to directly interact with Fyn kinase, one of the members of the

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212 non-receptor protein kinase family, resulting in an inhibition of the enzyme activity

213 [20]. It was also reported that the neuroprotective properties of the caffeic acid

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214 including anxiolytic effects, and the caffeic acid derivatives also possessed anti-

215 oxidant effects [21].

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Maybe, these functions participated to reduce CDV replication, but how the
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217 caffeic acid inhibited CDV is needed to explore. The caffeic acid plus RBV possessed
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218 higher antiviral effect than caffeic acid or RBV individually, at the -1 h and 0 h PI

219 stage, but the possible mechanisms will require further studies.
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220 In conclusion, we demonstrated that the caffeic acid played antiviral activity
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221 against CDV infection in vero cells. Our findings indicated that the caffeic acid might

222 represent a promising candidate for the treatment of CD in the future.

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223 Acknowledgement
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224 This work was supported by the Special Fund for Agro-scientific Research in the
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225 Public Interest (201403051) and. National Natural Science Foundation of China (No.

226 31372470).

227 Competing interests

228 The authors declare that they have no competing interests.

229

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334 Figure Captions

335 Figure 1. Chemical structures of Caffeic acid (A) and RBV (B)

336 Figure 2. Cytoxicity of Caffeic acid, RBV, and Caffeic acid plus RBV on vero

337 cells. (A) Cells were treated with 12.5-200 µg/mL for 72h. The cytoxicity was

338 evaluated using MTT assay. The viability of non-treated cells was considered to be

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339 100%. (B) Cytoxicity of RBV. (C) Cytoxicity of Caffeic acid plus RBV and 4 %

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340 DMSO. Experiments were done in triplicate. The data were analyzed by one-way

341 ANOVA (Dunnett’s t-test).

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342 Figure 3. Inhibitory effects of caffeic acid and RBV at different stages. (A) The

343 virus titer was reduced, Caffeic acid as an inhibitor on CDV has been shown after the

344
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addition of 32-64 µg/mL at different times PI. (B) After the addition of 8-32 µg/mL of
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345 RBV at different times PI. The virus titer was examined. (C) The inhibitory effect of
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346 Caffeic acid plus RBV against CDV were examined at different times PI. Experiments

347 were done in triplicate, and all differences are statistically significant in relation to
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348 control (**P<0.01). The data were analyzed by one-way ANOVA (Dunnett’s t-test).
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349 Figure 4. Antiviral effect of caffeic acid against CDV replication in vero cells by

350 real-time RT-PCR at three different times PI. Viral growth was measured in the
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351 presence of different concentrations of Caffeic acid after 24, 48, and 72 h PI. Viral
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352 RNA was quantified by real time PCR and the total RNA is expressed in copies/µl.
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353 Experiments were done in triplicate, and all differences are statistically significant in

354 relation to control (**P<0.01). The data were analyzed by one-way ANOVA

355 (Dunnett’s t-test).

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359 Table

360 Table 1 Cytotoxicity, anti-CDV activity and selectivity indices of the tested compounds.

IC50(µg/ml) SI
Compounds CC50(µg/ml)
-1 h 0h 1h 2h -1 h 0h 1h 2h

Caffeic acid >200 — — 23.3±0.9 32.3±0.7 — — >8.6 >6.2

RBV >200 — — 12.2±0.4 16.1±1 — — >16.4 >12.4

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361 a CC50 and IC50 values represent means ± SD.

362 b SI：Selectivity index （CC50/IC50）.

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363 c Stages of CDV replicative cycle: pre-treatment (1 h), adsorption (0 h), penetration (1 h), and intracellular (2 h).

364 The values are expressed as the means ± SD of the CC50 and IC50 values after at least three independent

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365 experiments.

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Highlights

1. The antiviral activity of the caffeic acid against CDV was

evaluated in vero cells.

2. The caffeic acid inhibits viral RNA accumulation during CDV

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replication cycle.

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3. The caffeic acid plus RBV inhibited the viral intracellular and

extracellular stage of replicative cycle.

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