Cells, virus and fusion protein
Swine testis cells (ST, CRL-1746) were purchased from ATCC and propagated in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10 % fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin. Porcine alveolar macrophage cells (3D4/21, CRL-2843) were a gift from Dr. Guoqing Shao (Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences) and were propagated in RPMI 1640 Medium supplemented with 10 % FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and Non-Essential Amino Acids (NEAA, Gibco). CSFV Shimen strain was purchased from China Institute of Veterinary Drugs Control. PTD-poMx1 fusion protein was expressed in E. coli and purified, denatured and refolded as previously described [17]. Aliquots of purified protein were stored at −80 °C.
Confocal microscopy
To determine the protein transduction efficiency, internalization of PTD-poMx1 was assessed by confocal microscopy. Briefly, ST and 3D4/21 cells grown on glass coverslips in 6-well plates were treated with 80 μg/ml PTD-poMx1 for 6, 12 and 24 h. Cells were then fixed with 4 % paraformaldehyde in PBS, and permeabilized with 0.2 % Triton X-100. Cells were incubated with anti-poMx1 mAb (1:500, ab79609, Abcam, USA) as a primary antibody for 1 h at 37 °C. After washing three times with PBS, cells were incubated with FITC-labeled goat anti-mouse IgG (1:200, ab150113, Abcam, USA) as a secondary antibody for 30 min at 37 °C. Cell nuclei were counter stained with 4’, 6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired by LSM710 confocal laser microscopy (Carl Zeiss Microimaging, Oberkochen, Germany), and processed with Photoshop software.
Western blot analysis
The transduction efficiency and stability of PTD-poMx1 in ST and 3D4/21 cell lines were evaluated by Western blot analysis as described previously [17]. Briefly, cells were cultured as exponentially growing subconfluent monolayers on 6-well plates. 80 μg/ml PTD-poMx1 was added to the cells at 37 °C. Every hour from 1 to 6 h, cells were washed three times with PBS and lysed in cold lysis buffer (1 % Triton X-100, 1 mM PMSF in PBS) for 10 min. Lysates were clarified by centrifugation at 12,000 x g for 10 min. Total cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membrane. PTD-poMx1 that translocated through the plasma membrane [22] and accumulated in the cytoplasm [17] was detected by anti-poMx1 mAb (1:1000). To assess the stability of PTD-poMx1 in the cells, 80 μg/ml PTD-poMx1 was added to the cells for 6 h at 37 °C. After washing three times with PBS, cells in DMEM free from PTD-poMx1 were incubated for a further 4, 8, 12, 16, and 20 h. Intracellular PTD-poMx1 in the cell lysates was detected by anti-poMx1 mAb (1:1000) as previously described. β-actin, a loading control, was detected by anti-β-actin antibody (1:5000, No. A5441, Sigma-Aldrich, USA).
Flow cytometry
To determine if PTD-poMx1 affects CSFV entry into host cells, CSFV adsorption at the cell surface was examined after treatment with PTD-poMx1 using flow cytometry and the data were analyzed with Cell Quest Pro software (Becton Dickinson). Briefly, ST or 3D4/21 cells treated with or without 80 μg/ml PTD-poMx1 were inoculated with CSFV at a MOI of 5 for 1 h on ice. The infected cell suspensions were prepared with PBS containing 0.02 % ethylene-diaminetetraacetic acid (EDTA), and incubated with the anti-E2 monoclonal antibody WH303 (1:100, Veterinary Laboratories Agency, Surrey, UK) for 1 h at 37 °C. After extensive wash, cells were treated with FITC-labeled goat anti-mouse IgG (1:200) at 37 °C for 30 min. Fluorescent signals on the cell surface were examined by a flow cytometer (Becton-Dickinson) and the percentage of positive cells was counted among 3 × 104 cells. Untreated cells were used as a negative control.
Quantitative real-time RT-PCR (qRT-PCR)
Viral genome replication was measured by qRT-PCR as previously described with modifications [21, 23]. Briefly, viral RNA was extracted from each sample using TRIzol reagent (Invitrogen, CA, USA). RNA pellets were suspended in 20 μl DEPC-treated water and a RT reaction was performed utilizing a RT reaction kit (Takara, Dalian, China). Target primers for the NS5B gene [24] and reference primers for the GAPDH gene [25] were used to quantify CSFV RNA. qPCR was carried out with SYBR Green PCR master mix according to the manufacturer’s protocol (Takara, Dalian, China). The data were analyzed by the 2-△△Ct method, and expression of the target gene was normalized to GAPDH mRNA levels in the same samples [26].
In vitro anti-CSFV activity of PTD-poMx1
Initially, three experiments were performed in parallel to investigate the antiviral activity of PTD-poMx1 against CSFV in ST and 3D4/21 cells. In the first experiment (treatment), cells (1.2 × 106) in 6-well plates were inoculated with CSFV at a MOI of 0.01 for 2 h at 37 °C. Cells were washed three times with PBS and maintained in medium containing 80 μg/ml PTD-poMx1 for 48 h. In the second experiment (prophylaxis), 80 μg/ml PTD-poMx1 was added to the cells (1.2 × 106) for 6 h at 37 °C. Cells were washed three times with PBS, then inoculated with CSFV at a MOI of 0.01 for 2 h at 37 °C. Cells were washed with PBS and maintained in medium free with PTD-poMx1 for 48 h at 37 °C. In the third experiment, 80 μg/ml PTD-poMx1 was inoculated simultaneously with CSFV at a MOI of 0.01 in the cells for 2 h at 37 °C. Cells were washed three times with PBS and maintained in medium free with PTD-poMx1 for 48 h at 37 °C. At 48 h post infection (hpi), viruses within the cells were released by freezing and thawing three times, and qRT-PCR was performed to quantify viral replication.
Animal experiment design
Nine four-week-old specific-pathogen-free Large White pigs were randomly assigned to three groups of three animals housed in three separate rooms. Prior to experiments, all pigs were examined serologically negative for certain important pathogenic viruses, such as CSFV, PRRSV, PCV2, FMDV, PRV, etc., using a series of commercial ELISA diagnostic kits from IDEXX Laboratories, Inc. and JBT Agency. Furthermore, all pigs were examined pathogenic negative for the tested viruses using a series of PCR/RT-PCR assays (data not shown). All animals were oro-nasally challenged with 105TCID50 CSFV Shimen strain. On the first day post challenge (1dpc), pigs in two groups (designated as 1 dose and 3 doses group, respectively) were injected with 1 mg/dose PTD-poMx1 protein via the ear vein. Pigs in the negative control group (designated as NC group) received equivalent saline via the ear vein. Pigs in 3 doses group were injected two additional times with 1 mg PTD-poMx1 at 3 and 5 dpc. Rectal temperature, clinical scores (1 point- no fever; 2 points- pyrexia + mild clinical signs; 3 points- severe clinical signs, and 4 points- death) and pathological lesions were monitored daily as previously described [27]. Blood samples (sera and EDTA-anticoagulated blood) were collected at 0, 3, 5, 7, 11, 14, and 21 dpc. White blood cell (WBC) counts were measured using an Abacus Junior Vet 5 (Diatron Group, Budapest,Hungary). Care of laboratory animals and animal experimentation were performed in accordance with animal ethics guidelines and approved protocols. All animal experiments were approved by the Animal Ethics Committee of Nanjing Agricultural University, and performed in High Technology Innovation Center of Animal Disease Control, Ministry of Agriculture, PR China (BSL-3 condition).
Viremia detection
Two experiments were performed to evaluate viremia. First, progeny viruses in sera were assessed using a TCID50 assay as previously described [28]. Second, viral RNA was extracted from whole blood using the RNeasy mini kit (Qiagen, Courtaboeuf, France), and viral load was detected using qRT-PCR.
Enzyme-linked immunosorbent assay (ELISA)
Two ELISA assays were performed to detect anti-poMx1 antibody or anti-CSFV-E2 antibody in sera as previously described with some modifications [29]. First, 96-well flat-bottom plates were coated with purified PTD-poMx1 at a concentration of 100 ng/well in coating buffer (Na2CO3/NaHCO3, pH 9.6) at 4 °C overnight. Plates were washed 3 times with PBST, blocked with 1 % BSA in PBST at 37 °C for 2 h, and then washed 3 times with PBST. 100 μl pig sera (1:100) was added to each well. After incubation at 37 °C for 1 h, the plates were washed 3 times with PBST and then incubated with HRP-labeled goat anti-pig IgG antibody (1:5000, sc-2463, Santa Cruz, USA) at 37 °C for 1 h. After the final wash, 100 μl of fresh TMB substrate (Sigma-Aldrich, USA) was added per well and plates were incubated for 10 min. The reaction was stopped by 2 M H2SO4, and optical density (OD) was measured at 450 nm using ELISA plate reader (ELX800). Second, the antibodies to CSFV E2 in the sera were assessed using the IDEXX CSFV Antibody Test Kit (IDEXX Laboratories, Inc., Maine, USA) according to the manufacturer’s protocol.
Statistical Analysis
All data are presented as means ± standard deviation (S.D.) as indicated. Student’s t-test was used to compare pairs of treated or untreated groups. Statistical significance is indicated as not significant (ns) (P > 0.05), * (P < 0.05), and ** (P < 0.01). All statistical analyses and calculations were performed using GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA).