5.1 Virus, cells and reagents
The RB-1B strain of MDV is cell-associated virus, and it was stored in our laboratory in liquid nitrogen. Chicken embryo fibroblast (CEF) cells were prepared from 9 days old SPF chicken embryo. The SPF chicken embryos were obtained from the Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China), and the animal experiments were performed according to the institutional animal care guidelines and approved by the Animal Care Committee of College of Veterinary Medicine, Yangzhou University. The fully confluent monolayers of CEF cells were grown in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Shanghai China) supplemented with 5% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37°C in a 5% CO2 atmosphere. Baicalin was purchased from SIGMA (Shanghai, China) and diluted in dimethyl sulphoxide (DMSO). The specific monoclonal antibody against MDV gB protein, BA4, was generated in our laboratory. And the anti-tubulin monoclonal antibody was purchased from SIGMA (Shanghai, China).
5.2 Cytotoxicity test
The cytotoxicity of baicalin on CEF cells was determined using a cell counting kit, CCK-8 kit (Beyotime, Shanghai, China). Briefly, 4.0×104/well CEF cells were seeded in 96-well plates, and 150 μL of baicalin at 0, 5, 10, 20 and 40 μg/mL in DMEM maintenance medium was added to each well. After incubating for 72 hours in a 37°C incubator, CCK-8 solution (15μL) was added to each well. Incubation was continued for 1 hour and the absorbance at 450 nm was measured. The relative cell viability rate was determined as a percentage for each concentration as (OD450 drug)/(OD450 control)×100.
5.3 Virus infection and chemical treatment
To investigate the inhibitory effect of baicalin on MDV replication, CEF cells were seeded in 12-well plates (6.0×105 cells/well) and pretreated for 2 hours at 37°C with DMEM maintenance medium containing 0.5% serum and 0, 2 or 20 μg/mL baicalin. The cells were then infected with 200 PFU of MDV and incubated under the exposure of the drug for 96 h before harvesting. The collected cells were used for plaque counting, real-time PCR, western blotting and indirect immunofluorescence assay.
To determine the dynamics of baicalin inhibition of viral replication, CEF cells in 12 well plates were pretreated with 20 μg/mL baicalin for 2 hours, and then 200 PFU of MDV per well was inoculated. After 4 hours of virus adsorption, the medium was replaced by 20 μg/mL baicalin diluted in DMEM maintenance solution with 0.5% serum . Virus infected cells were harvested at 1, 2, 3, 4 and 5 days respectively for plaque counting, real-time PCR detection and indirect immunofluorescence assay.
5.4 Different models of drug treatment and virus infection
In order to investigate the mechanism of viral inhibition by baicalin, a time-of-drug addition experiment was carried out as described previously with minor modifications [11]. Briefly, in the first protocol (P1), CEF cells were pre-treated with 20 μg/mL baicalin for 2 hours at 37°C before viral adsorption. After removing the culture medium containing the drug, the cells were inoculated with 200 pfu virus and incubated with the maintainance medium containing 0.5% FBS at 37°C in an atmosphere of 5% CO2 until harvest; in the second protocol (P2), a mixture of 200 PFU virus suspension and 20 μg/mL of baicalin were added to cell monolayers and incubated for 2 h at 37°C. After removing the medium, the cells were incubated with maintainance medium at 37°C in an atmosphere of 5% CO2; in the third protocol (P3), 20 μg/mL of baicalin was added to maintainance medium after virus adsorbing for 4 hours in a 37°C incubator. After 96 hours of incubation, viral gene expression and plaque formation were examined for all protocols. In order to understand the correlation between baicalin and virus infection, the time course analysis was performed. CEF cells were infected with 200 PFU MDV for viral adsorption. Baicalin (20 μg/mL) was then added into the cells at 0 h, 1 h, 6 h, 12 h, and 24 h after virus adsorption, respectively. At 96 hours post infection (p.i.), cells were haversted for viral gene expression and plaque formation.
5.5 Virucidal assay
200 pfu virus were mixed with 20 μg/mL of baicalin and incubated at 37°C for 1.5 hours. After centrifugation to remove the supernatant, the virus-infected cells were resuspended and added to CEF in a 12-well plate. The inoculum was replaced with fresh 0.5% serum containing maintenance medium after 4 hours of virus adsorption. The cells were harvested after 96 h post infection for plaque counting and real-time PCR.
5.6 Plaque counting
Virus infected CEF cells in 12-well plate were digested with 400 μL 0.05% trypsin per well. 100 μL per well of 10 fold serial diluted cells were added into 96-well CEF cells with 12 replicates of each dilution The cells were incubated at 37°C in an atmosphere of 5% CO2. The number of viral plaques was counted after 96 hours. The highest dilution which has plaques in all 12 wells was used for titer calculation. The formula used is PFU/mL = (total plaques/12) × 10 × dilution factor.
5.7 Quantitative reverse transcriptase polymerase chain reaction
The expression levels of the viral genes and cytokines were determined with real-time PCR (7500 Real-Time PCR System, ABI) as previously reported [21] . The sequences of the primers are provided in Table 1, and these primers were synthesized by Gene Script Company (Nanjing, China).
Total RNA from CEF cells was prepared using the AxyPrep Multisource Total RNA Miniprep Kit (AXYGEN, USA). 1 μg of RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, USA). The expression levels of viral genes were determined by real-time SYBR green quantitative PCR (7500 Real-Time PCR System, ABI). The diluted cDNA (1 μL), 400 nM primer and 10 μL of SYBR Green Master Mix were used for real-time PCR in a total volume of 20 μL reaction. The amplification conditions were as follows: 95°C for 30 seconds, then 40 cycles, 95°C for 5 seconds and 60°C for 34 seconds. A dissociation curve was generated to analyze each PCR product after 40 cycles. The analysis of relative gene expression data was performed using the 2-ΔΔCT method with the chicken 18S as the internal reference gene.
5.8 Indirect immunofluorescence
The infected cells were fixed with acetone ethanol (3:2) for 7 minutes at room temperature. After washing three times with PBS, the fixed cells were incubated with anti-gB mAb BA4 (5 μg/mL) in PBS for 60 min at 37°C, then washed with PBS followed by incubation with goat anti-mouse IgG conjugated with FITC (Sigma, USA) at room temperature for an additional 30 min. The pictures were captured with a OLYMPUS fluorescence microscope.
5.9 Western blot analysis
Similar to our previous report [11], after cell lysing with RIPA buffer, the protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad, USA). The proteins (30 μg) were denatured by heating (5 min, 100 °C) and electrophoretically separated in 12% SDS-PAGE under reduction conditions. The protein was then transferred onto a nitrocellulose membrane (Sigma, Shanghai, China). The membrane was blocked with 5% skimmed milk containing PBST (PBS containing 0.1% Tween 20) for 1 hour and incubated with the anti-gB monoclonal antibody BA4 (10μg/mL) and anti-tubulin antibody (10μg/mL) as internal control for 2 hours at room temperature. The membrane was washed three times with PBST and incubated with the appropriate HRP secondary antibody (SIGMA, Shanghai, China) for 60 minutes at 37°C. After washing with PBST, the membrane was developed using Bio-Rad enhanced chemiluminescent substrate and photographed with an ultrasensitive chemiluminescence detector (Proteinsimple, USA).
5.10 Statistical analyses
The results represent the means ± standard deviations (SD) of triplicate determinations. The significance of the variability between the trials was analyzed using GraphPad (version 5.0) software. Differences between samples were assessed by the Student’s t-test, and p values <0.05 were statistically significant. The experiment was performed at least three times independently with the similar results.