Reagents and materials
SVV-LNSY01-2017 used in this study was isolated from the pig with Porcine primary vesicular disease (PIVD). Baby hamster kidney cells (BHK21 cells; ATCC, CCL-10) were cultured in Dulbecco’s modified essential medium (HyClone, SH30022.01) containing 10% fetal bovine serum (Gibco, 16000044) and 100 U/ml of penicillin (GENVIEW, GA3502) at 37°C in a humidified 5% CO2 incubator. Myeloma cells SP2/0 (ATCC, CRL-1581) were grown in RPMI 1640 medium (HyClone, SH30809.01) containing 10% FBS, 100 U/ml of penicillin grown under the same conditions as those described above. SPF level BalB/c mice were purchased from the Animal Experimental Center of Huazhong Agricultural University. Protein A/G Agarose (sc-2003) was purchased from Santa Cruz Biotechnology. Alexa Fluor 555 goat anti-mouse (A32727) antibodies were obtained from Invitrogen. Polyvinylidene fluoride (PVDF, 46978100) membranes were purchased from Roche.
Virus propagation and purification
BHK-21 cells were infected with an amount of 0.01 MOI SVV-LNSY01-2017 when the cell culture area reached 80%. BHK-21 cells with typical cytopathic effects (CPE) were collected at 48 h post-infection (hpi) and then repeatedly frozen and thawed 2 times, and then the virus solution was harvested. The virus solution was concentrated and precipitated at 30,000 rotating speed per minute (r/min), and then the supernatant was discarded and the precipitation was resuspended with PBS (pH = 7.4). After dissolved at 4 °C for 12 h, the supernatant was collected by centrifugation at 12,000 rpm and then centrifuged at 40,000 rpm by a sucrose gradient. Subsequently, the virus band was taken up for de-sucrose treatment, and the precipitation was resuspended in PBS (pH = 7.4) and stored at -80°C. Purified viruses were further identified by SDS-PAGE and western blot analysis.
Monoclonal antibody production
4-week-old female SPF BalB/c mice purchased from the Laboratory Animal Center of Huazhong Agricultural University (Wuhan, China), were subcutaneously immunized with 40 μg of purified SVV-LNSY01-2017 particles with an equal amount of complete Freund's adjuvant. After the initial immunization, mice were immunized 2 times with the same dose of purified SVV-LNSY01-2017 particles with an equal amount of incomplete Freund's adjuvant at two-week intervals. After mice were immunized three times, serum samples were collected from the immunized mice and serum titers were detected by indirect ELISA/virus neutralization assay. Three days after the fourth booster immunization, mice splenocytes were harvested and fused with SP2/0 using 50% polyethylene glycol (50% PEG, Sigma, Aldrich). Hybridoma culture supernatants were screened by virus neutralization assay and the positive hybridoma cells were cloned by a limiting dilution. Stable hybridoma clones (106 cells) were injected intraperitoneally into liquid paraffin-pretreated abdominal cavities of BalB/c mice. Then the monoclonal antibodies were harvested and purified by a Protein A/G purification kit (NAbTMProtein A/G Spin Kit, Thermo Scientific, Fremont, CA, USA), and their activity was characterized by western blot and indirect immunofluorescence assay (IFA).
Virus neutralization assay
The antibody-secreting hybridoma cells are screened by virus neutralization assay. Briefly, the hybridoma cell supernatant was serially diluted 2-fold, mixed with 200 TCID50 SVV-LNSY01-2017 in equal volumes, and placed in a 37°C CO2 incubator for 2 h. The mixture was inoculated into a single layer of BHK-21 cells in a 96-well plate at 100 μL/well, and cultured for 3 to 4 days in a CO2 incubator at 37°C. Then the cytopathic condition was recorded to identify neutralizing hybridoma cells. The neutralizing hybridoma cells were subcloned into new 96-well plates by limiting dilution. After three rounds of subcloning, the hybridoma cells capable of stably secreting neutralizing antibodies were obtained.
Indirect enzyme-linked immunosorbent assay
ELISA plates were coated overnight at 4°C with 100 μL of inactivated SVV-LNSY01-2017 (1 μg/mL) diluted in bicarbonate coating buffer (1.59 g/L Na2CO3 and 2.93 g/L NaHCO3, pH = 9.6). The wells of the plate were washed 3 times with PBS and then blocked with 5.0 % bovine serum albumin (BSA) in PBS (PBSA) for 2 h at 37°C. The wells were drained and incubated with 100 μL of two-fold serially diluted mAbs (from 1:100 to 1:12800) for 1 h at 37°C. The wells were then washed three times with PBS containing 0.05% Tween-20 (PBST) and then incubated with 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10000, Boster, Wuhan, China) for 1 h at 37°C. After 3 times washing with PBST, the reactions were developed with 50 μL/well substrate A (0.1 M citrate/phosphate buffer [pH 5.0]) and 50 μL/well solution B (0.04% o-phenylenediamine; 0.14% H2O2) for 10 min at room temperature and then terminated with 50 μL/well of 2 M H2SO4. The optical densities (OD) at 630 nm were measured using a microplate reader.
Western blot identification of monoclonal antibodies
293T cells were transfected with plasmids to express the indicated protein. The 5×loading buffer and the protein sample were uniformly mixed at a ratio of 1:4, boiled at 95℃ for 10 min, and immediately placed on ice to cool. After loading in an amount of 10 μL per well, SDS-PAGE electrophoresis was carried out, and the protein on the polyacrylamide gel was transferred to the PVDF membrane by semi-drying. The membrane was blocked with 5% skim milk at room temperature for 2 h; the supernatant of the hybridoma cells was added (1:5), and incubated for 2 h at room temperature. The membrane was washed 5 times with PBST, and then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5000) for 1 h. The positive bands were visualized by electrochemiluminescence (ECL) reagents and developed on film.
Indirect immunofluorescence identification
BHK-21 cells were cultured in 96-well plates. When the cells are full of a single layer, the cells were inoculated with 2000 TCID50 SVV-LNSY01-2017. When the cell cytopathic effect reached 70%~80%, it was fixed with 4% paraformaldehyde for 30 min permeated with 0.2% Triton X-100 at room temperature for 20 min. The cells were blocked with PBS (pH = 7.4) containing 1% BSA for 30 min and then washed with PBS three times. 100 μL/well of hybridoma cell supernatant was added to different culture wells and the samples were incubated at 37℃ for 1 h. After washed with PBS three times, Alexa Fluor 555 goat anti-mouse antibodies (1:1000 dilution) were added and the samples were incubated for 30 minutes in dark with room temperature. After washing three times, the results were observed under a fluorescence microscope.
Construction of mutant infectious SVV cDNA clones
Infectious SVV cDNA clones were conserved in our laboratory. Mutagenesis of SVV-E157A construct was performed using site-directed mutagenesis (C214-01/02; Vazyme). The construct was identified through DNA sequencing. To rescue viruses, plasmids containing full-length viral cDNAs were transfected into 293T cells using JetPRIME (PT-114-07; Polyplus Transfection) according to the manufacturer’s protocol. When about 70% of cells exhibited CPE, the media was collected. Then the virus was serially passaged five times on BHK-21 cells. Stocks from each passage were stored at -80˚C. Each passage viruses were sequenced to identify the stability of virus.
The preceding experiments were performed in triplicate. The various treatments were compared by an unpaired, two-tailed Student's t-test while assuming unequal variance. P < 0.05 was considered statistically significant. Meanwhile, P < 0.001 and P < 0.0001 were marked with two (**) and three (***) asterisks, respectively.