Virus, cells, plasmids, and animals

JEV (strain P3) was stored at − 80 °C. It was used as the coating antigen and stimulus for in vitro experiments and used for challenge experiments.

Vero cells were used for plasmid transfection and plaque assays to detect viral titers, and the plaque reduction neutralization test (PRNT) was used to detect the nAb titers. C6/36 cells are used for virus proliferation assays.

The pV-JP3ME plasmid was constructed by introducing the BamHI enzyme digestion site, Kozak sequence and signal sequence upstream of the prM/E sequence of the P3 strain and introducing the XhoI digestion site downstream into the eukaryotic expression vector pV.

Specific pathogen-free 6- to 8-week-old female BALB/c mice were used for immunity, sera and splenocyte collection and challenge tests. The results presented are from a single experiment or are from three independent experiments.

Reagents and instruments

The restriction enzymes BamHI and XhoI, eukaryotic expression vector pV, nuclear staining agent 4′,6-diamidino-2-phenylindole (DAPI), and transfection reagent Lipofectamine 3000 were purchased from Thermo Scientific (USA). Minimal essential medium (MEM) and RPMI-1640 medium were purchased from Gibco (USA). Methylcellulose was purchased from Sigma (USA). Goat anti-mouse fluorescein isothiocyanate (FITC)-IgG antibody was purchased from Beijing TransGen Biotech (China). Goat anti-mouse horseradish peroxidase (HRP)-IgG antibody was purchased from Abcam (USA). Tetramethylbenzidine (TMB) substrate color solution was purchased from MabTech Company (USA). The enzyme-linked immunospot (ELISPOT) kit, streptavidin and AEC color development kit were purchased from BD Company (USA). The gene introduction instrument was purchased from Shanghai Teresa Corporation (China). The enzyme-linked immunosorbent assay (ELISA) plate reader and cell culture incubator were purchased from Thermo (USA). The ELISPOT plate reader was purchased from CTL (USA).

Transfection and immunofluorescence experiments

pV-JP3ME or pV was transfected into Vero cells. After 5 h, the transfection plasmid/reagent mixture was discarded and replaced with complete culture medium. After 40 h, the medium was discarded, and the two groups of cells were simultaneously fixed. Then, the cells were incubated with JEV antiserum (1:1000) as the primary antibody and goat anti-mouse FITC-IgG as the secondary antibody. Observation of specific green fluorescence under a fluorescence microscope indicates that the plasmid was successfully transfected and expressed in mammalian cells in vitro. Fluorescence microscope imaging was performed at 200× magnification, and the microscope was from Nikon, Japan.

Animal experiments

The mice were randomly divided into two groups. The vaccine group was vaccinated with the DNA vaccine pV-JP3ME, and the control group was vaccinated with the empty vector plasmid pV. Immunization was performed three times, and each immunization dose was 50 μg. Intramuscular injection (i.m.) with electroporation (EP) was used. One and 3 weeks after the last immunization, the splenocytes and sera of the two groups of mice were collected, respectively. The next day, mice in the vaccine and the control group were challenged with JEV. The body weight changes and the survival rate were measured daily after the challenge, and the observation continued for 12 consecutive days. The animal experiment schedule is shown in Fig. 1.

Fig. 1

Mouse experimental workflow. Groups of mice were immunized by intramuscular electroporation with 50 μg of either the pV-JP3ME DNA vaccine or pV in each limb individually and were boosted twice at three-week intervals. Splenocytes were obtained 1 week after the final immunization, and sera were collected 3 weeks after the final immunization. Subsequently, the vaccinated mice were challenged with 1 × 105 PFU of the JEV P3 strain. The body weight changes and the survival rates were observed for 12 consecutive days after the challenge

Plaque assay

Vero cells were cultured in 24-well plates, and the cell density was more than 95% before virus infection. The serially diluted virus was serially diluted 10-fold from the original solution (1:1) for a total of seven dilutions, namely, 1:1 to 1:106. Two hundred microliters of the virus dilution was added to each well and incubated at 37 °C for 1 h. The plate was gently shaken once every 15 min. After the dilution was discarded, 5 mL of MEM medium containing 1.2% methylcellulose was added to each well. After 4 d of culture, the medium was discarded, and 1 mL of crystal violet staining solution was added to each well and stained for 30 min at room temperature. The number of plaques per well was counted, and the virus titer was calculated. The titer of the virus solution was repeatedly measured three times, and an average value was taken and is expressed as the plaque-forming unit (PFU)/mL.


Heat-inactivated JEV was coated in a 96-well plate at 105 PFU per well at 4 °C overnight. The coating antigen was discarded and blocked with 1% bovine serum albumin (BSA) at 37 °C for 2 h. The blocking solution was discarded. The sera of each group of mice were started from 1:100 and were serially diluted at a twofold ratio for a total of 12 dilutions, namely, from 1:100 to 1:204,800, and added to the wells in turn as a primary antibody at 4 °C overnight. The next day, the primary antibody was discarded. After the plate was washed five times, HRP-labeled goat anti-mouse IgG antibody (1:4000) was added as a secondary antibody. After incubation at 37 °C for 1 h, the plate was discarded. The substrate solution developed color for 20 min, and the reaction was stopped with H2SO4. We used 1/2 of the A450 nm value at the 1:100 dilution of the control group as the cut-off value, and the maximum dilution greater than this cut-off value is the serum IgG antibody titer.


Vero cells were cultured in 24-well plates as described previously. The serum was diluted from 1:10 and serially diluted at a 2-fold ratio. There are seven consecutive dilutions, that is, 1:10 to 1:640. Each dilution of serum was mixed with an equal volume of virus solution (containing 100 PFU). For incubation at 37 °C for 1 h, serum-free virus samples were set at 4 °C and 37 °C to exclude temperature factors and reference positive virus counts. Then, the serum/virus mixture was added to the wells in order and incubated at 37 °C for 1 h. During the period, the plate was shaken gently every 15 min. The subsequent step was the same as described in 2.5. The serum dilution corresponding to a 50% reduction in the number of plaques in the positive wells incubated at 37 °C was recorded as the PRNT50 value, which is the nAb titer.


IL-2 and IFN-γ capture antibodies diluted 1:200 were coated in a 96-well plate at 4 °C overnight. We discarded the coating solution and blocked with RPMI-1640 medium containing 10% FBS at room temperature for 2 h. Then, the splenocytes from the two groups were added to each well at 2 × 105 cells per well, and 105 PFU heat-inactivated JEV was added as a stimulus and cultured at 37 °C for 72 h. After the cultured splenocytes were discarded, IL-2 and IFN-γ detection antibodies were added, and then, the spot forming units (SFUs) were determined by adding streptavidin and AEC chromogenic solution.

Statistical analysis

All experimental data were recorded using Excel 2016 software, statistical analysis was performed using SPSS 17.0 software (USA), the body weight change was analyzed by repeated analysis of multivariate analysis of variance, survival rates were compared using the log-rank test, and the differences between the groups were compared using one-way ANOVA. Quantitative data are expressed as the mean ± standard deviation. P < 0.05 was considered statistically significant.

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