Epithelioma papulosum cyprini (EPC) cells were cultured in medium 199 (M199) (Hyclone, USA), supplemented with 10% fetal bovine serum (FBS) (Every Green, China), penicillin (100 IU/mL), and streptomycin (0.1 mg/mL) (Beyotime, China) and maintained at 25 °C.
The SVCV strain (0504) was isolated from common carp (Chen et al., 2006) and kindly provided by Prof. Qiang Li (Key Laboratory of Mariculture, Agriculture Ministry, PRC, Dalian Ocean University, Dalian, China). The strain was then propagated in EPC cells containing M199 supplemented with 2% FBS until a cytopathic effect (CPE) was observed. Subsequently, the virus was harvested and stored at –80 °C until further use. The virus was titrated into 96-well plates and 50% tissue culture infective dose (TCID50) analysis was performed according to the Reed-Muench method (Pizzi, 1950).
Due to their easy susceptibility to SVCV infection (Encinas et al., 2013), adult zebrafish (Danio rerio) are considered an ideal experimental model for studying the antiviral effect of medicines on SVCV in vivo (Liu et al., 2019; Shen et al., 2018; Shen et al., 2019; Qiu et al., 2020). Zebrafish (total length 3.17±0.24 cm, body weight 0.61±0.23 g, mean±SD) were purchased from the Beilun Aquatic Breeding Center (Ningbo, China), maintained in a static water system consisting of four 200 L aquarium ponds at 20 °C, and fed commercial fresh blood worms thrice per day. Ten zebrafish were randomly selected to verify SVCV-free status, as described previously (Koutná et al., 2003). Prior to in vivo tests, experimental fish were transferred into new containers and acclimatized at 15 °C for one week. All experiments are performed according to the Experimental Animal Management Law of China and approved by the Animal Ethics Committee of Ningbo University.
The detailed synthesis route of C10 is shown in Figure 1. Briefly, 7-hydroxycoumarin (8.1 g, 50 mmol) in acetone (150.0 mL) was added to anhydrous K2CO3 (13.8 g, 100.0 mmol) and KI (200.0 mg) with stirring for 30 min at room temperature. Afterwards, 1,6-dibromotexane (14.6 g, 60.0 mmol) was mixed into the reaction and refluxed at 60 °C for 24 h. The precipitate was then filtered and washed with acetone (400.0 mL), and the organic layer was combined and concentrated. Purification was performed via silica gel column chromatography with mixed petroleum ether and ethyl acetate (5:1, v/v) as the eluent, resulting in compound 1,7-(6-bromohexyloxy) coumarin (8.9 g) (yield 54.6%). To synthesize compound 2, a mixture containing 7-(6-bromohexyloxy) coumarin (650.6 mg, 2.0 mmol) with benzimidazole (472.6 mg, 4.0 mmol) and anhydrous K2CO3 (1.4 g, 10.0 mmol) was stirred at room temperature for 24 h. After filtering and evaporation, the organic layer was dried and concentrated. The residue was purified using silica gel chromatography with chloroform/methanol (10:1) as the eluent to obtain compound 2 (i.e., C10) as a white solid (529.3 mg) with a yield of 73.1%. C10 was dissolved in dimethyl sulfoxide (DMSO) and used at the indicated concentrations.
Cell viability assays were performed with a Cell Counting Kit-8 (CCK-8, Beyotime, China), which used WST-8, a water-soluble tetrazolium dye, to enhance sensitivity of the WST-8-based assay over conventional 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Briefly, serial dilutions of C10 in M199 supplemented with 2% FBS, at concentrations of 0.4 mg/L to 25.0 mg/L (in triplicate), were added to EPC cells in 96-well plates. After every 24 h, cell viability assays using CCK-8 were performed as per the manufacturer’s protocols. After 2 h of incubation at 37 °C, colorimetric absorbance from the mitochondrial enzyme substrate reaction was measured at 450 nm using a microplate reader (ELX800, Gene, Hong Kong, China). As DMSO exhibits no cytotoxicity at doses up to 0.1% (v/v) (Liu et al., 2020), signals were compared to solvent-treated cells (DMSO up to 0.05%, v/v).
Antiviral activity assays were performed as per previous research (Liu et al., 2017). Briefly, EPC cells seeded on 12-well and 96-well plates were infected with SVCV at 103×TCID50/mL at 25 °C, followed by the addition of serial dilutions of C10. After 48 h of infection, the antiviral activity of C10 against SVCV replication in EPC cells was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and CCK-8.
The sampled cells were harvested and maintained in 2.5% glutaraldehyde at 4 °C overnight. The sampled cells were prepared for scanning (SEM) and transmission electron microscopy (TEM) as described in previous studies (Liu et al., 2015; Zhang et al., 2011). Cells were observed using SEM (Hitachi, Japan) and ultrathin sections were observed using TEM (1200EX TEM, JEOL, Japan).
EPC cells in 12-well plates were incubated with SVCV for 1 h at 25 °C to allow the virus to both attach to and enter cells. The virus was removed, and cells were washed twice with M199 medium before the addition of fresh media. At various viral replication time points (i.e., 0, 2, 4, 6, 8, 12, 18, and 24 h post-infection (hpi)), up to 12.5 mg/L C10 was introduced to assess its effects on SVCV infection under time-of-addition study. For time-of-removal, SVCV-infected cells were exposed to 12.5 mg/L C10 at 0 hpi. At the same time points as above, the medium containing C10 was replaced with cell culture maintenance medium. Infected cell cultures were treated with 0.025% DMSO as the control and all samples were harvested at 24 hpi, and the related expression of SVCV protein N was analyzed by RT-qPCR. Triplicate wells were used for each treatment at each time point.
EPC cells were incubated with SVCV (103×TCID50/mL) and 12.5 mg/L C10 at 25 °C for 48 h. After incubation, the cells were washed thrice with phosphate-buffered saline (PBS) and treated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (1 mg/L) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (5 mg/mL). Fluorescence signals were monitored using upright fluorescence microscopy (NI-U; Nikon, Japan). Additionally, apoptosis of the sampled cells was enumerated using a mitochondrial membrane potential assay kit with 5,5',6,6'-tetrachloro-1, 1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1) (Beyotime, China) according to the manufacturer’s instructions. As an ideal fluorescent probe, JC-1 generates a matrix of mitochondrion-forming aggregates that produce a red color when ΔΨm levels are high, whereas lower ΔΨm transforms JC-1 to its monomeric form, resulting in a green color. Fluorescence intensity was measured using a MACSQuant 10 Analyzer, and a minimum of 10 000 cells were counted in each treatment.
We used 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) oxidant-sensitive probes to assess intracellular ROS levels. Briefly, EPC cells seeded on 12-well plates with coverslips were infected with SVCV in the presence of C10 at a concentration of 12.5 mg/L. The samples of SVCV-infected and C10-treated cells were harvested and incubated with 10 μmol/L DCFH-DA and 5 μmol/L DHE following the instructions provided in the kits (Beyotime, China). Fluorescence signals were monitored using an upright fluorescence microscope (NI-U; Nikon, Japan).
Zebrafish were randomly selected and separated into four aquaria containing UV-sterilized water. The rearing temperature of each aquarium was maintained at 15 °C. The experimental fish were intraperitoneally injected with 20 μL 104×TCID50/mL SVCV. After 12 h, SVCV-infected fish were injected intraperitoneally with 50 mg/L C10 to test survival over an additional 7 d, and thus calculate mortality. The DMSO vehicle control was set at 0.1% (final concentration). To avoid deterioration of water quality, dead fish were immediately removed from the water.
Under the same operational procedures, three virus-infected and C10-treated fish were collected at 24 h, 48 h, and 96 h, and frozen at –80 °C until RNA and protein extraction. Exposure was conducted in static water with aeration. The fish were humanely euthanized with 40 μg/mL (final concentration) tricaine methanesulfonate (MS-222). The sampled fish were thawed and homogenized with 10 volumes of M199 using a stomacher or manual homogenization under ice-bath cooling. The homogenates were subjected to low-speed centrifugation at 2 000 g for 10 min at 4 °C. The virion in the supernatants was filtered through 0.45 μm membranes (Millipore, USA). The viral loads were detected using TCID50 analysis.
The harvested cell and tissue samples were immediately extracted with cold PBS (0.1 mol/L, pH 7.4) in a cool ice-bath. The homogenates were centrifuged at 10 000 g for 15 min at 4 °C to obtain supernatants. Total protein content of the supernatants was determined using the Bradford method (Bradford, 1976) with bovine serum albumin (Beyotime Institute of Biotechnology, China). Four common antioxidant indices, i.e., glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA), were measured using commercial assay kits (Jiancheng Institute and Beyotime, China) according to the manufacturer’s instructions. All measurements were recorded on a microplate reader.
The samples were collected and immediately frozen in liquid nitrogen for subsequent RNA isolation. Total RNA was extracted using TRIZOL reagent (Takara, Japan) following the manufacturer’s protocols, and subsequently eluted in 10–40 μL nuclease-free water for storage at –80 °C. Nucleic acid concentrations were quantified using a NanoDrop spectrophotometer (ThermoFisher, USA). Extracted RNA purity was determined using the A260nm/A280nm ratio with absorbance at 260 and 280 nm between 1.8 and 2.0, respectively. A total of 500 ng/μL of purified RNA was used for cDNA generation per reaction, which was reverse transcribed using HiScript Q Select RT SuperMix for qPCR (Vazyme, China). RT-qPCR was performed using ChamQTM SYBR® qPCR Green Master Mix (Vazyme, China) in an ABI StepOnePlusTM Real-Time PCR Detection System (ThermoFisher, USA). PCR analysis was amplified as follows: 95 °C for 30 s, denaturation with 40 cycles at 95 °C for 15 s, and annealing at 60 °C for 60 s. To assess the specificity of each amplicon, melt curve analysis was also performed at the end of each thermal profile. The primer sequences are listed in Table 1. Each individual sample was run in triplicate.
Gene Primer sequence (from 5' to 3') EPC-actin Forward GCTATGTGGCTCTTGACTTCGA Reverse CCGTCAGGCAGCTCATAGCT SVCV glycoprotein (G) Forward GCTACATCGCATTCCTTTTGC Reverse GCTGAATTACAGGTTGCCATGAT SVCV nucleoprotein (N) Forward AACAGCGCGTCTTACATGC Reverse CTAAGGCGTAAGCCATCAGC SVCV phosphoprotein (P) Forward TGAGGAGGAATGGGAATCAG Reverse AGCTGACTGTCGGGAGATGT SVCV matrix protein (M) Forward ATTCGGTCAAATGCCTCCTT Reverse GCCTATCTTTTCCCCGTTTA Fish-18S Forward ACCACCCACAGAATCGAGAAA Reverse GCCTGCGGCTTAATTTGACT ISG15 Forward ACTCGGTGGTGATGCTCCTC Reverse CCTTCGGCACTCTCTCTTTC IFNγ Forward ATGATTGCGCAACACATGAT Reverse ATCTTTCAGGATTCGCAGGA RIG-I Forward TTGAGGAGCTGCATGAACAC Reverse CCGCTTGAATCTCCTCAGAC MHC-II Forward ATCTGCTAAAACTTTTTCTTGCC Reverse GAACCCTACACACTTCACCTCTG aoc2 Forward GCATAAAGATGAAGAGCAGACCA Reverse ATGTGTAGGAAACCAGCAGTGAC Mx Forward ATAGGAGACCAAAGCTCGGGAAAG Reverse ATTCTCCCATGCCACCTATCTTGG
Table 1. Sequences of primer pairs used for analysis of gene expression by RT-qPCR
Relative mRNA expression levels were calculated using the 2−△△Ct method with the formula △△Ct=(Ct, target gene–Ct, reference gene)–(Ct, target gene–Ct, reference gene)control (Livak & Schmittgen, 2001). Drug response curves were represented by a logistic sigmoidal function with a maximal effect level (Amax) and Hill coefficient representing the sigmoidal transition (Origin 8.1, USA). Virus titers were log10 transformed prior to statistical analyses. For the experiments, unpaired Student’s t-tests were used to compare significant differences between drug-treated and negative-control DMSO samples (SPSS 18.0, USA), presented as mean±SD. A Kaplan-Meier test was used for the survival rate of zebrafish. P-values of less than 0.05 were considered statistically significant.
Hydroxycoumarin efficiently inhibits spring viraemia of carp virus infection in vitro and in vivo
- Received Date: 2020-02-10
- Accepted Date: 2020-04-24
- Available Online: 2020-05-10
- Publish Date: 2020-07-01
- Antiviral effect /
- Spring viraemia of carp virus /
- Interferon response /
- Antioxidant-oxidant balance
Abstract: Spring viremia of carp virus (SVCV) causes devastating losses in aquaculture. Coumarin has an advantageous structure for the design of novel antiviral agents with high affinity and specificity. In this study, we evaluated a hydroxycoumarin medicine, i.e., 7-(6-benzimidazole) coumarin (C10), regarding its anti-SVCV effects in vitro and in vivo. Results showed that up to 12.5 mg/L C10 significantly inhibited SVCV replication in the epithelioma papulosum cyprini (EPC) cell line, with a maximum inhibitory rate of >97%. Furthermore, C10 significantly reduced cell death and relieved cellular morphological damage in SVCV-infected cells. Decreased mitochondrial membrane potential (ΔΨm) also suggested that C10 not only protected mitochondria, but also reduced apoptosis in SVCV-infected cells. For in vivo studies, intraperitoneal injection of C10 resulted in an anti-SVCV effect and substantially enhanced the survival rate of virus-infected zebrafish. Furthermore, C10 significantly enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain antioxidant-oxidant balance within the host, thereby contributing to inhibition of SVCV replication. The up-regulation of six interferon (IFN)-related genes also demonstrated that C10 indirectly activated IFNs for the clearance of SVCV in zebrafish. This was beneficial for the continuous maintenance of antiviral effects because of the low viral loads in fish. Thus, C10 is suggested as a therapeutic agent with great potential against SVCV infection in aquaculture.
|Citation:||Lei Liu, Da-Wei Song, Guang-Lu Liu, Li-Peng Shan, Tian-Xiu Qiu, Jiong Chen. Hydroxycoumarin efficiently inhibits spring viraemia of carp virus infection in vitro and in vivo. Zoological Research, 2020, 41(4): 395-409. doi: 10.24272/j.issn.2095-8137.2020.037|