Cells and viruses
Marc-145 cells (China Center for Type Culture Collection, China), an immortalized cell line derived from African green monkey kidney cells, were cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning, USA) containing 10% fetal bovine serum (FBS) (PAN, Germany), which are permissive to PRRSV replication and are commonly used in laboratories. PRRSV strain CHR6 (Classical North American type PRRSV strain) was provided by Dr. Guihong Zhang from South China Agricultural University, and PRRSV-EGFP, a recombinant virus showing growth replication characteristics similar to those of the wild-type virus in the infected cells, was gifted by Dr. Shuqi Xiao from Northwest A&F University. CHR6 and PRRSV-EGFP were used to infect Marc-145 cells. The virus strains were propagated in Marc-145 cells and titrated as 50% tissue culture infective dose (TCID50).
The cytotoxicity of TPP was detected with the alamarBlue® assay (Invitrogen, USA) according to the manufacturer's instructions. Marc-145 cells (1 × 104/well) were seeded in 96- well plates, different concentrations of TPP were added in DMEM medium when cells grew to 60-70% confluence. After incubation for 48 h in Marc-145 cells, 10 μL of alamarBlue® was added to each well, and incubated for another 3 h. At last, the fluorescence value was detected using Multi-Mode Reader (Synergy2, BioTek, USA) at the absorbance of 570 nm.
Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR)
To detect the relative expression of PRRSV ORF7 and cytokines, qRT-PCR was performed. RNA was extracted from cultured cells using TRIzol reagent (Magen, China). Reverse Transcription System (A3500, Promega, USA) was used for reverse transcription in 20 μL reaction volume following the manufacturer's instructions. The reverse-transcription primers were Oligo (dT) 15 primer (C110A, Promega, USA) and Random primer (C118A, Promega, USA). Reverse transcription products were amplified by a LightCycler 480 Real-Time PCR System (LC480, Roche, Switzerland) using 2 × RealStar Green Power Mixture (GenStar, China). The primers are listed in Table 1. qRT-PCR reaction system was run under the following conditions: 95 °C for 10 min, then 95 °C for 15 s, 60 °C for 1 min and 72 °C for 30 s went through 40 cycles, finally 72 °C for 10 min. Data were normalized to GAPDH in each individual sample. The 2-ΔΔCt method was used to calculate relative expression changes. Relative expression (fold changes) was compared to mock infected cells.
Six-well-plate cell samples (2 × 106/well) were harvested in cell lysis buffer (Beyotime, China) containing PMSF (Beyotime, China). Processed samples were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinyl difluoride (PVDF) membrane (Roche, USA). Then the membranes were blocked with 5% BSA (Ruishu, China) in TBST (20 Mm Tris-HCl PH8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at 37 °C. After blocking they were incubated with an anti-PRRSV N protein monoclonal antibody (1:1000 dilution, Jeno Biotech, Inc., Republic of Korea), and an anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (1:1000 dilution, Cell Signaling Technology, USA) overnight at 4 °C. After washing three times with TBST, membranes were incubated with an anti-mouse IgG, HRP-linked antibody or anti-rabbit IgG, HRP-linked antibody (1:1000 dilution, Cell Signaling Technology, USA) for 1 h at 37 °C. The antibody signals were exposed using an enhanced chemiluminescence (ECL) reagent (Fdbio Science, China).
Cells were seeded in six-well plates and grown to 70 - 80% confluence. There were two approaches to analyze the antiviral effect of TPP. (I) Pre-treatment: Cells were pre-treated with different concentrations of TPP (0, 50 and 100 μg/ml) for 2 h, PRRSV-CHR6 was then added and cultured for 36 h. (II) Post-treatment: Cells were inoculated with PRRSV-CHR6 for 4 h, then the inoculum was removed and TPP (0, 50 and100 μg/ml) was added to cells for 36 h.
Viral attachment, entry, replication and release assays
For attachment assay, cells were cooled for 2 h at 4 °C, and then infected with PRRSV-CHR6 at a multiplicity of infection (MOI) of 0.6 in the presence of different concentrations of TPP (0, 25 and 50 μg/ml) for 2 h at 4 °C. After rinsing with cold PBS three times, cells were replenished with fresh DMEM containing 2% FBS for 24 h at 37 °C. The cells were collected for western blot analysis so that we could determine the effect of TPP on viral attachment. As for entry assay, cells were inoculated with PRRSV-CHR6 (MOI = 0.6) for 2 h at 4 °C. After binding to the cell surface, cells were washed with PBS three times and cultured at 37 °C for 2 or 4 h in the presence of various concentrations of TPP (0, 25, and 50 μg/mL). Cells were then washed with PBS and incubated for another 24 h at 37 °C. The cells were collected for western blot analysis so that we could determine the effect of TPP on viral internalization. As for replication assay, cells were inoculated with PRRSV-CHR6 (MOI = 0.6) at 37 °C for 6 h, then various concentrations of TPP (0, 25, and 50 μg/mL) were added for 2 or 4 h. Cells were then washed with PBS and incubated for an additional 24 h at 37 °C. The cells were collected for western blot analysis so that we could determine the effect of TPP on viral replication. For release assay, cells were incubated with PRRSV-CHR6 (MOI = 0.6) for 24 h at 37 °C. After that, cells were rinsed with PBS three times and TPP at different concentrations was added to the cells for 3 h at 37 °C. At last, cells were collected for western blot analysis to detect the PRRSV N protein expression.
Immunofluorescence Assay (IFA)
Cells were fixed by 4% paraformaldehyde for 15 min. After permeabilized with 0.5% Triton X-100 for 15 min at room temperature (RT), cells were blocked with 1% bovine serum albumin (BSA) for 30 min and then incubated with a rabbit monoclonal antibody against the p65-protein (1:500 dilution, Cell Signaling Technology) or an antibody against PRRSV nsp2 (a gift from Dr. Hanchun Yang, China Agricultural University, 1:1000) at 4 °C overnight. Cells were then incubated with an anti-rabbit secondary antibody conjugated with Alexa Fluor® 555 or 488 (Cell Signaling Technology, MA, USA) at 1:1000 dilution for 2 h. Nuclei were stained using DAPI (1:1000; Cell Signaling Technology). Cells were examined by fluorescence microscopy (Nikon, Japan).
All experiments were performed with at least three independent replicates. Student's t-test and one-way ANOVA were used to analyze the data. Statistical analysis was performed using SPSS 17.0 and GraphPad Prism 6.0. P < .05 was considered to be significant.