Construction and the design of PEDV-S transfer bacmids
The full-length gene sequence of S protein of PEDV Pintung 52 strain passage five (PEDV-PT; GenBank Accession No. KY929405) were codon optimized (GenBank Accession No. MN586852) for the insect protein expression system and synthesized (ProTech, Taipei, Taiwan) as previously described (Chang et al. 2018a). In attempt to deliver the interest gene to the BmN cells, the gene of full-length S was cloned into pBPxhE transfer vector (pBPxhE-S-Bm), following the suggested protocol of the In-Fusion® HD Cloning Kit (Clontech Laboratories Inc., Fremont, CA, USA) (Chang et al. 2012). The pBPxhE-S-Bm transfer vector contains a polyhedrin (polh) promoter of BmNPV, a viral GP64 signal peptide, and the 6× His tag that drive gene expression, lead protein synthesis, and label the target protein (Fig. 1). The plasmid also has an enhanced green fluorescent protein (EGFP) which driven by a Drosophila heat-shock 70 (Hsp) promoter as a reporter fluorescence in the BmN cell and mammalian cells.
Construction and viral titer determination of PEDV S displaying BmNPV
The recombinant BmNPV viral DNA, namely vBmpDsRFP, containing a Bsu36I restriction enzyme recognition site and tagged with a red fluorescent protein (RFP), was linearized by the digestion of Bsu36I restriction enzyme (NEB, Ipswich, MA, USA) under 37 °C for 1 h, then co-transfected with the PEDV-S transfer vector, the pBPxhE-S-Bm, to the BmN cells by using the TransIT®-Insect Transfection Reagent (Mirus, Madison, WI, USA). Five days after the transfection, to obtain the recombinant S-Bm virus, the cells were tenfold serial diluted in culture medium to perform the limited dilution for selecting a BmN cell exhibiting both EGFP (derived from pBPxhE-S-Bm) and REP (derived from vBmpDsRFP) positive. The viral titers of each clone were determined by the evaluations of 50% tissue culture infection dose per milliliter (TCID50/mL) (LaBarre and Lowy 2001). Briefly, the BmN cells (Bombyx mori ovary cell line, ATCC No. CRL-8910™) were cultured in TC 100 insect medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco) at 26 °C and seeded in the 96 well plates with the total cell count of 4 × 104. After 1 h incubation, each clone of the recombinant virus was serially diluted and applied onto the BmN cells with eight duplications. Following a 30-min centrifugation at 2000 rpm in order to promoting the viral infectivity, these plates were incubated at 26 °C for 4 to 5 days. The cytopathic effect (CPE) was observed and the viral titer of each clone were determined. The recombinant virus, namely S-Bm, which had a highest viral titer of 108 TCID50/mL, was selected for the further characterizations and analysis. The S-Bm was propagated in the BmN cells and store at 4 °C until use.
Detection of PEDV S expression in BmN cells by immunofluorescence assay (IFA)
The BmN cells in 80% confluency were inoculated with 10 multiplicity of infection (MOI) of the S-Bm and harvested 3 days after the inoculation. In order to detect the S protein expression in the BmN cells, the immunofluorescence assay (IFA) was performed. Briefly, the cells were fixed on the plates by using 4% paraformaldehyde (Sigma, MO, USA). After blocking with 3% BSA (Sigma, MO, USA) for 1 h and following by three times of PBS washes, the anti-PEDV S specific monoclonal antibody generated in our previous study (Chang et al. 2019), namely P4B-1, was applied on the cells for 1 h. Following three times washing with PBS, the goat anti-mouse IgG conjugated with Alexa Fluor 555 (Invitrogen, CA, USA) was applied onto the cells for 1 h. After washing with PBS, the cells were mounted with the Hoechst mounting solutions (Thermo Fisher Scientific, Waltham, MA, USA) to depict the nuclei. The result of florescence was observed under microscope.
Expression and detection of the PEDV S in silkworm pupae
The pupae of the OJ03 × OJ04 strain Bombyx mori kindly provided by the Miaoli District Agricultural Research and Extension Station were directly injected with a total of 4 × 104 TCID50 S-Bm in the volume of 100 μL. In the control group, the larva and the pupae were reared separately and injected with 4 × 104 TCID50 wild-type BmNPV in the volume of 100 μL. Four days after the inoculation, the signal of EGFP fluorescence of the silkworm was observed under UV irradiation.
Western blotting
To determine the protein expression level, the S-Bm infected BmN cells and the homogenized S-Bm infected pupae were collected. The BmN cells were collected 3 days after the S-Bm inoculation and lysed with the RIPA (Thermo Fisher Scientific, Waltham, MA, USA) buffer. Meanwhile, the S-Bm pupae were collected 4 days after inoculation. The hard puparium of pupae was removed, and the naked body of the pupae was homogenized by Bullet Blender Tissue Homogenizer (Next Advance Inc., NY, USA) with 1.5 mL lysis buffer containing 1 M Tris–HCl (Merck, Darmstadt, Germany), 0.5 M EDTA (Merck), 5 M NaCl (Merck), 10% (w/v) Brij96 (Merck), 10% (w/v) NP40 (Merck), 0.01% formalin (Merck) and sodium azide (Merck) (Chang et al. 2012). After centrifuge at 6000 rpm for 30 min, the supernatant of the homogenized sample was collected for the denaturation. The samples from the BmN cells and the homogenized pupae were denatured by boiling the samples under 95 °C for 10 min with the Laemmli Sample Buffer (Biotools, New Taipei city, Taiwan). The samples were then subjected to the electrophoresis in the gradient sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (PAGE) gel (HR gradient gel solution, TOOLS, Taiwan) and then transferred to the PVDF membrane (Millipore, Darmstadt, Germany). The membrane containing protein samples was washed briefly in PBS (Omics bio, Taipei, Taiwan) and blocked by 5% skim milk for an hour at room temperature. Then, the mouse anti-6× His-tag monoclonal antibody (1:5000 dilution, EnoGene, NY, USA) was utilized as the primary antibody to probe the target proteins. Following by three times washing with PBS, the goat anti-mouse IgG antibody conjugated with HRP (1:5000 dilution, Invitrogen) was used as the secondary antibody for the signal detection. After three times washing, the signals were detected by using the Clarity™ Western ECL Blotting Substrates (Bio-Rad, CA, USA) using the Classic Blue Autoradiography film BX (Life Science, MO, USA).
Immunization program of piglets
The 4-week old, crossbred Large White × Duroc piglets were obtained from a conventional farm with no G2b PEDV outbreak history. Twenty piglets confirmed to be PEDV-seronegative by a PEDV S-specific ELISA were selected and used in this animal study. These piglets were separated into four groups according to the different treatments: the S-Bm cell group (n = 5), the control group (n = 5), the S-Bm pupae group (n = 5), and the WT-Bm pupae group (n = 5). As shown in Fig. 2, All animals were treated three times at 2 weeks interval. The piglets in the S-Bm cell group were orally fed with 2 × 106 S-Bm infected BmN cells, which contained 12 μg recombinant S protein, in 5 mL TC 100 insect medium at 0, 2, and 4 week post first vaccination (WPFV); while the piglets in the control group were orally fed with 5 mL TC 100 insect medium. Each piglet in the S-Bm pupae and WT-Bm pupae group were orally feed with 6.5 g S-Bm-infected silkworm pupae containing 100 μg recombinant S protein or non-infected silkworm pupae at 0 and 2 WPFV, and fed with 40 g silkworm pupae containing 615 μg recombinant S protein at 4 WPFV. Piglets in different groups were housed in different rooms and all the pigs were labeled with ear tags. Ten milliliter blood samples in 1 mL 5% (w/v) EDTA anti-coagulated buffer and oral swabs were collected on day 0, day 13, day 27, and day 41 post priming for evaluating PEDV S-specific systemic IgG or mucosal IgA. The plasma of the blood samples was stored under − 20 °C until use. The oral swabs were re-suspended in 1 mL of PBS (Gibco, Gaithersburg, MD, USA) and also stored under − 20 °C until use.
PEDV S-specific ELISA for detecting systemic IgG and oral mucosal IgA
The PEDV S-specific ELISA for detecting the systemic IgG and oral mucosal IgA of pigs was established and conducted as previous described with some modifications (Chang et al. 2018b). Briefly, the purified S protein of PEDV expressed by HEK 293 cells were diluted and coated on the Nunc maxi-soap plates (Thermo Fisher Scientific, Waltham, MA, USA) in the concentration of 2 ng/μL. After a 16-h incubation under 4 °C, the plates were washed six times with washing buffer (KPL, SeraCare, Milford, MA, USA) in the volume of 200 μL, then followed by an hour of blocking procedure by incubating the plates with blocking buffer (KPL, SeraCare) under room temperature. For detecting systemic PEDV specific IgG, plasma of piglets as well as a positive serum control and a negative serum control were 40-fold diluted in the blocking buffer (KPL, SeraCare) and applied onto the plates for an hour incubation. To detect the oral mucosal IgA level of piglets, the re-suspended supernatant of the oral swabs was twofold diluted in the blocking buffer (KPL, SeraCare) and applied onto the PEDV S coated plates for 16-h incubation under 4 °C. Then, the plates were washed three times with washing buffer and incubated with 1000-fold diluted secondary antibodies, the horseradish peroxidase (HRP)-conjugated goat anti-pig IgG (KPL, SeraCare) or a HRP-conjugated goat anti-pig IgA antibody (KPL, SeraCare) in the dilution of 5000-fold for another 1 h. After the plates were completely washed six times with wash buffer (KPL, SeraCare), the ABTS® Peroxidase Substrates (KPL, SeraCare) was added onto the plates in the volume of 50 μL and incubated for 3 min under room temperature. To stop the coloration reaction, 50 μL stopping solution (KPL, SeraCare) was added. The plates were read by the EMax Plus Microplate Reader (Molecular Devices, Crawley, UK) under the wavelength of 405 nm to obtain the optical density (OD) values of each well. The value of positive control comes from a PEDV-hyperimmune pig, while the value of negative control comes from a seronegative piglet.
Statistical analysis
The results of systemic IgG and IgA level were statistically analyzed by statistical analysis system version 9.4 (SAS 9.4, SAS Institute Inc., Cary, NC, USA) and compared by one-way analysis of variance (ANOVA). The significance was determined to have a p value < 0.05 (p < 0.05).