Cells, viruses, and plasmids
BHK-21(Baby hamster kidney clone 13 cells; strain 21; ATCC CCL-10) were maintained and propagated in minimal essential medium (MEM) with 10% fetal bovine serum (FBS, Gibco, Australia). BSR-T7/5 cells expressing T7RNA polymerase were obtained from Karl-Klaus Conzelmann (Max-von-Pettenkofer Institut, Munich, Germany) and were cultivated in Glasgow minimal essential medium (GMEM) supplemented containing 10% FBS and 4% tryptose phosphate broth, 10% FBS. Besides, in alternate passages, 1 mg/ml G418 was added to ensure maintenance of the T7 polymerase gene. All cells were grown in a humidified chamber at 37℃ supplemented with 5% CO2. The plasmid pO/DY-VP1, a previously constructed type O FMDV full-length infectious clone which based on the constructed FMDV vaccine strain pOZK/93 − 08 , 3B1 and 3B2 were mutated(4AGP6-4TAA6) to eliminate the dominant epitope of 3B non-structural protein, served as the genetic backbone to construct some recombinant cDNA clones containing single or multiple capsid-stabilizing mutations in the structural protein. The virus recovered from pO/DY-VP1 is referred to as rHN/DY-VP1 (parental virus) which cannot react with monoclonal antibody 3B4B1.
Identification of stabilizing residues and Generation of recombinant cDNA mutants
Some recombinant virus mutants were designed in the plasmid pO/DY-VP1 (Fig. 1a) for studying the function of single or multiple mutations. According to the reported substitutions responsible for the thermostability [20,21], three mutants S2093F, S2093H and Y2098F were introduced in VP2. Additionally, V2090A was included in subsequent process because this substitution was suspected to help other introduced mutant for being passaged stably in cells. Five schemes were designed, containing S2093F, Y2098F, V2090A-S2093F, V2090A-S2093H and V2090A-S2093H-Y2098F. The site-directed mutagenesis were performed by a bio-tech company called GENEWIZ (Suzhou, China, www.genewiz.com.cn) and all the molecular constructs were performed by using standard molecular biological techniques . All recombinant plasmids were verified by nucleotide sequencing and ensure that there was no other mutation occurring in the cloning process.
Transfection and virus recovery
The Not I-linearized recombinant plasmids were purified by a QIAquick PCR Purification Kit (Qiagen), and transfected into BSR/T7 cells monolayers with a Gene Pulser Xcell (two pulses at 150V and 950 uF) and Ingenio® Electroporation Kits (Mirus). The cells were further maintained at 37℃ in growth medium. After 48–72 h, The BSR-T7 cells showed cytopathic effects (CPE) and were frozen at -80℃. After thawing and centrifugation, the virus-containing cell culture supernatants (named ‘passage 0’, P0) were serially passaged 8 times on BHK-21 cells. The total RNA was extracted from passage 8 supernatant of each rescued virus, and nucleotide sequencing verified that the mutated viruses derived from the genome-length cDNA (Fig. 1a).
Plaque, growth kinetics and thermal inactivation assays
Each plaque assays were performed in a 6-well plate and repeated 3 times. BHK-21 monolayer cells were infected with 200uL serial dilutions of viral samples. After 1 h incubation at 37°C, the cells were overlaid with 2 mL gum tragacanth (MP Biomedicals). After incubating at 37°C for 48 h, cells were fixed in 50% acetone and 50% methylalcohol and stained with 1% crystal violet, as previously described .
Growth kinetics of the virus was performed with plaque assays. Confluent monolayers of BHK-21 cells were infected with mutant viruses and parental virus at a multiplicity of infection (MOI) of 0.1 at 37°C. After 1h of adsorption, the inoculum was removed, and cells were washed with 0.01 M phosphate-buffered saline (PBS; pH7.4) to remove unattached viruses. Then, cells were supplemented 2mL complete medium and incubated at 37°C. At 4, 8, 12 and 20 h.p.i., the virus-infected supernatants were collected and frozen at -80°C. Virus titers were calculated by plaque-forming units (PFU) mL− 1, as described elsewhere .
Thermal inactivation was performed on parental and recombinant viruses cell culture supernatants which were uniformly diluted to 5×105 PFU/mL in TNE buffer (100 mM Tris, pH 7.4, 10 mM EDTA, 150 mM NaCl), completely as described previously . After that, infectious particles were put into 42℃ water bath for different times of heat treatment. To be specific, all viruses were incubated at temperatures of 42°C for 0, 15, 30, 45, 60, 120, 180, 240 min and 49°C for 0, 15, 30, 45, 60 min. Immediately after the heat treatment, these viruses were cooled onto the pre-prepared ice, and the viruses were titrated on BHK-21 cells by plaque assays. Each experiment was repeated twice. In addition, a pH inactivation kinetics assay was performed in TNE buffer (pH = 6.0) with a steady temperature (25°C) for 0, 15, 30, 45, 60, 120 min. The logarithmic values of all viruses titers at the different time points were linearly fitted respectively, then the slopes were calculated which could be used to determine the inactivation rate [27,28]. The percentage of infectious particles remaining was also computed and plotted , which was determined as the titer at each time point divided by the initial titer (0 min)×100%.
Virus inactivation, Purification
The eighth passages of parental and mutants’ viruses were expanded and cultured on BHK-21 cells. After being freezed-thawed for 3 times, the cells were centrifuged at 4℃ at 1500 × g for 30min to remove the cell fragments and collect the supernatant and lysates. Clarified supernatant was made 1.2% in binary ethyleneimine (BEI) and inactivated at 30℃ for 28h . During the inactivation period, the mixture is reversed and mixed per hour. Inactivated virus was inoculated into 2-day-old suckling mice to test whether the virus was inactivated completely .
Inactivated viruses were concentrated with 80 mL/L polyethylene glycol 8000 (PEG 8000; Sigma-Aldrich) and purified on 10 g/mL to 50 g/mL sucrose density gradients (SDG), and the mixture was centrifuged at 4℃, 104 000 × g for 3 h. Following fractionation, 146S were collected by measuring the absorbance at 260 nm. The collected 146S was centrifuged at 130 000 × g for 4h at 4℃ to remove sucrose, and the supernatant was discarded. The precipitation was then resuspended with 200 uL TNE buffer (50 mM Tris/HCL [pH 7.4], 10 mM EDTA, 150 mM NaCl) .
BHK-21 cells (2 ×105) grown on a six-well plate were infected with parental and mutant virus at a MOI of 1 respectively. At 5 h post-infection (p.i.), the cells were fixed with 4% paraformaldehyde for 30 min at 4℃. The cells were washed three times and permeabilized for 10 min with 0.5% Triton X-100 in PBS and blocked for 1 h with 5 g/mL BAS bovine serum albumin in PBS. Then, the cells were washed with PBS 3 times and incubated for 1 h with MAb 3A24 or 3B4B1 (MAb 3A24 directed against AEKNPLE (residues 99–105) epitope in NSP(Non-SP) 3A of FMDV and MAb 3B4B1 directed against GPYAGPMER (residues 1–9) epitope in NSP 3B2 were obtained from the Lanzhou Veterinary Research Institute (LVRI)), then the cells were washed 5 times and stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (purchased from Sigma) for another 1h. The cells were observed in a Leica DMI6000B fluorescence microscope.
The animal experiments were performed under Biosafety Level 3 conditions in the animal facilities at LVRI following the protocol approved by the Review Board of LVRI, Chinese Academy of Agricultural Sciences (Permission number: SYXK-GAN-2004-0005). At the beginning of the experiments, all animals were negative for FMDV specific antibodies. The animals were euthanized by intravenous injection of sodium pentobarbital at the end of all experiments.
Briefly, sixteen 3-month-old pigs were divided into 4 groups which were kept in a separate room respectively. Pigs in group 1 were inoculated with the vaccine containing inactivated parental virus, and which in group 2–4 were inoculated with the vaccine containing inactivated mutant virus separately. Each animal received 3µg of purified 146S antigen formulated in water-in-oil-in-water emulsions with Montanide ISA 201 (Seppic, Paris, France) by intramuscular injecting on days 0. Furthermore, serum samples collected at 7, 14, 21, 28, 35 and 42dpi were tested by virus neutralizing-antibody titers (VNT) as previously described .
For the guinea-pig experiments, inactivated parental and stabilized mutant viruses were emulsified with ISA201 adjuvant, then 40 guinea pigs, aged 2 months, were divided into 4 groups (n = 10 per group). Group 1 was inoculated with the vaccine which contained inactivated parental virus and was stored for 4 months at 4°C, and group 2–4 was inoculated with the vaccines which contained inactivated mutant virus respectively and were stored for 4 months at 4°C. Each guinea pig received 0.5 µg of purified 146S antigen by intramuscular injection. Furthermore, serum samples collected at 28 dpi were tested by virus neutralizing-antibody titers (VNT) as previously described . The experiments were repeated with the same volumes after the vaccines had been stored for 6 months at 4°C.
No statistical method was used to predetermine sample size. Group sizes for the pigs and guinea-pigs immunization studies were consistent with those in previously published studies [33,34].
Titration of neutralizing antibodies
The sera from guinea pigs and pigs were prepared from blood samples. Neutralizing-antibody titers were calculated by the Spearmann-Karber method, and were expressed as the reciprocal of the highest serum dilution neutralizing 50% of 100 TCID50 of the homologous virus . The mean antibody titers of each group were compared with threshold antibody titers that have previously been shown to correlate with protection [33,34].
All statistical analyses and the graphs were carried out using GraphPad Prism v5.0 (GraphPad Software). P < 0.05 was considered statistically significant.