Open access peer-reviewed chapter
Abstract
Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide. Climate change and the spread of mosquitoes are contributing to the virus’s geographical expansion into previously unaffected regions. Traditional virus quantitation techniques and methods for measuring neutralizing antibodies to JEV, such as the plaque assay, are considered gold standards. However, the plaque assay is labor intensive, low throughput, and time-consuming. The recent interest in developing new JEV vaccines necessitates a rapid, high-throughput, and sensitive technique to assess vaccine effectiveness. Here, we provide a high-throughput viral quantitation and neutralizing antibody measurement system based on the focus forming assay (FFA). This assay has a high throughput, is comparable to the plaque assay, and is as sensitive as quantitative real-time RT-PCR in detecting low virus particle counts. We further validated the assay to measure neutralizing antibodies to JEV in patients who have recovered from Japanese encephalitis. This approach enables the rapid and high-throughput quantification of JEV-neutralizing antibodies, making it valuable for vaccine evaluation.
Keywords
- Japanese encephalitis virus (JEV)
- JEV neutralizing antibodies
- focus forming assay (FFA)
- focus reduction neutralization test (FRNT)
- plaque assay
- qRT-PCR
- JE vaccine
1. Introduction
JEV is one of the leading cause of viral encephalitis and is a significant health concern in tropical and subtropical regions. This vector borne virus transmitted by Culex species of mosquito belongs to Flaviviridae family, consisting of several other medically important virus like Yellow Fever virus, Dengue virus, Zika virus, West Nile virus, tick-borne encephalitis virus, Murray Valley encephalitis virus, Saint Louis encephalitis virus, etc. [1, 2, 3, 4, 5, 6]. Based on nucleotide sequencing of the viral genome, JEV is divided into five different genotypes—GI, II, III, IV and V with each of them having a characteristic geographical distribution [7]. The transmission of this disease generally follows bird-mosquito-bird cycle with the amplifying hosts as pigs and human are the dead-end hosts [8]. A study on Honshu Island in Japan and many other studies has shown a basic cyclic pattern of transmission of virus in birds, mosquito and swine, which would ultimately lead to an outbreak of disease [9, 10, 11]. Every year around 100,000 clinical cases are seen in Asian countries with case fatality rate as high as 30% and more than 60% survivors suffer from neurological sequelae [12]. There are no effective antiviral treatments for Japanese encephalitis. The treatment consists of supportive care with intravenous (IV) fluids and antipyretics [13, 14]. Vaccination is only the preventable measures and to date, around 15 vaccines are available globally against JE, based on genotype GIII. They are available mainly in four different types- cell culture derived live attenuated, mouse brain-derived killed inactivated, cell culture derived killed, inactivated and live attenuated recombinant (chimeric) vaccine. The SA14-14-2 live attenuated JE vaccine is the most extensively used vaccine across the countries among children and adults [15, 16, 17, 18, 19, 20].
The efficacy of the vaccine depends on generation of antibodies against the virus and suitable detection techniques are required in order to evaluate the vaccine. To check the neutralizing antibodies against JEV the most widely accepted method is plaque reduction neutralization test (PRNT) [21]. However this assay has few limitation as it is time-consuming, labor intensive and has low throughput. Although the mechanism through which the plaque assay and focus forming assay works is the same, the volume of sample it uses and the detection methods is different [22]. This makes the focus forming assay a reliable method as it uses the less volume of samples and give the result at shorter duration as compared to plaque assay. This method can also be used for the detection of neutralizing antibodies against several other arboviruses [23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33].
Here, we present a detailed protocol for the focus reduction neutralization test (FRNT) to quantify neutralizing antibodies against JEV. We also demonstrate the high-throughput nature of the FRNT by comparing its performance with the plaque assay and real-time PCR. Notably, FRNT demonstrated its effectiveness as a reliable conventional assay for detecting JEV-neutralizing antibodies, even within a short timeframe of 24 hours.
2. Results
2.1 Identification of an appropriate cell line and titration of various JEV genotypes using plaque assay
The optimal cell line for the plaque assay was identified by testing two distinct cell lines – the Vero cell line and the porcine stable kidney cell line (PS cell line). The cell culture-adapted Japanese encephalitis virus strain XZ0934 was used. The test was performed in 24-well-flat bottom cell culture plates. After a 72-hour incubation period post-infection, the cells were fixed and stained with crystal violet, and plaque enumeration was done using an ELIspot reader. Notably, the Vero cells exhibited small and unclear plaques in contrast to the PS cells, which displayed clear plaques with a gradual reduction in successive dilutions (Figure 1A). While the plaque quantification was not significantly different between the two cell lines, the PS cells were selected for virus quantitation in the plaque assay. Following the selection of the suitable cell line, various genotypes of cell culture-adapted JEV, including GV (XZ0934), GIII (P20778), and GIII (JaOArS982), were quantified (Figure 1B). It is evident from the results that the GV (XZ0934) strain exhibited higher viral titers. Consequently, the strain XZ0934 was selected for subsequent experiments.
Figure 1.
Selection of suitable cell line and titration of different JEV genotypes using plaque assay. (A). Plaque morphologies of JEV-XZ0934 in PS and Vero cell lines. Plaques were developed on a Vero and PS cell monolayer after 3 days of incubation. Statistics by unpaired t-test, n = 3. (B). Titration of Different genotypes of JEV in PS cell line monolayer after 3 days of incubation, n = 3.
2.2 Selection of suitable cell line and virus titration in focus forming assay
The Vero cell line and the porcine stable kidney cell line (PS cell line) were tested for their suitability for virus quantitation using a focus forming assay. The cell monolayer of two distinct cell lines was infected with JEV strain XZ0934. After 24 hrs of infection, the virus foci inside the cells were detected using a defined protocol, and the foci generated were counted in ELIspot reader (Figure 2A). Vero cells showed significantly higher foci count and titer than the PS cells (p = 0003), affirming the suitability of the Vero cell line for virus quantitation in focus forming assay (Figure 2B). To explore the effect of extended incubation on the viral titer estimation, the assay was also terminated at 48 hrs post-infection. The titers were not significantly different between 24 and 48 hours (Figure 2C, D). Additionally, the foci started merging when the incubation duration was extended, suggesting that the 24-hour time point suffices as a termination point for the focus forming assay.
Figure 2.
Selection of suitable cell line and titration of JEV XZ0934 using focus forming assay. (A). Representative image of foci morphology in PS and Vero cells, n = 3. (B). Foci were developed on a Vero and PS cell monolayer after 24 h of infection with JEV-GV strain (XZ0934). Statistics by unpaired t-test ***P ≤ 0.001, n = 3. (C). Representative image of FFA in Vero cell line at 24 and 48 hrs. (D). Foci were developed on Vero cells and compared between 24 and 48 hrs of termination after infection with JEV-GV (XZ0934). Statistics by unpaired t test, n = 3.
2.3 Comparative analysis between plaque assay and focus forming assay
The performance and the detectability were then compared between plaque and focus forming assay. Vero cells were seeded in a 96-well plate for the focus forming assay, while PS cells were seeded in a 24-well plate for the plaque assay. Serial dilutions of the JEV (XZ0934) were prepared and added to both plates. The focus forming assay was terminated at the 24-hour time point, while the plaque assay was terminated at 72 hours post-infection (Figure 3A, B). There was no significant difference found between the titers calculated using plaque assay and focus forming assays (Figure 3C). This finding implies that the focus forming assay provides results within 24 hours of infection, and these results exhibit titers that are comparable to those obtained through the golden standard plaque forming assay.
Figure 3.
Comparative analysis between plaque assay and focus forming assay. (A). Representative image of plaque morphology in PS cells. (B). Representative image of foci morphology in Vero cells. (C). Plaque and foci were developed at 72 h and 24 h post-infection with JEV (XZ0934). Statistics by unpaired t test, n = 2.
2.4 Comparative analysis between focus forming assay and real-time PCR
To assess the concordance between the actual viral titer and the titer determined through the focus forming assay (FFA), a comparative experiment was conducted between the focus forming assay and real-time PCR. Different dilutions of JEV-GV XZ0934 were used for the FFA, and from the same dilution, RNA was extracted using the Qiagen Viral RNA extraction kit. The FFA was executed following a standardized procedure, while the RNA was detected using real-time PCR. A standard plot was established using a JEV NS3 clone, and the unknown values were plotted (Figure 4A). To evaluate statistical significance, a correlation plot was generated (Figure 4B). The results indicated a positive correlation between the FFA-derived titer and that obtained through real-time PCR, confirming the sensitivity and accuracy of the FFA-derived titer.
Figure 4.
Comparative analysis between focus forming assay and real-time PCR. (A). Standard curve of Japanese encephalitis virus using JEV NS3 clone. (B). Spearmen correlation of copy number versus focus forming units, p = 0.0008, r2 = 0.9545.
2.5 Focus reduction neutralization test (FRNT) for Japanese encephalitis virus
The applicability of a high-throughput focus assay that could be completed within 24 h was tested using the JE samples. For this focus reduction neutralization assay was performed for JEV-infected patients (n = 10) samples using GIII (JaOArS982) and GV (XZ0934) strains. The experiment was carried out according to the mentioned protocol and terminated 24 hours after infection (Figure 5A). The quantitation of titer suggested the presence of neutralizing antibodies in all the patients, which was above the seroprotective titers (Figure 5B). However, a significant decline was observed in the antibody titers in the tested samples against JEV-GV strain as compared to the JEV-GIII strain (Figure 5B). These results demonstrate the applicability of the focus forming assay for quantitation of neutralizing antibodies against multiple strain and genotypes of JEV.
Figure 5.
Focus Reduction neutralization assay for Japanese encephalitis virus. (A). Representative image of focus reduction neutralization test of Japanese encephalitis in month 3 infection sample (Image: autoimmun diagnostika GMBH (AID) EliSpot reader machine). (B). FRNT graph plotted with Wilcoxon match-pair rank test. **P ≤ 0.002.
3. Materials and methods
3.1 Cell culture and virus
Vero cell line was maintained in Dulbecco’s modified Eagle medium (DMEM) medium containing 10% (v/v) inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Porcine stable kidney cell line (PS) was maintained in minimum essential medium (MEM) medium containing 10% (v/v) inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin.
3.2 Generation and purification of Japanese encephalitis virus
All procedures should be followed in BSL2 containment with appropriate safety training.
Materials:
Vero cells
JEV viral stock
Dulbecco’s modified Eagle medium (DMEM, high-glucose) containing 2% fetal bovine serum (FBS).
1X dulbecco’s phosphate-buffered saline (DPBS).
Tissue culture flasks.
50-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon).
Syringe and 0.22 μ syringe filter.
2 ml Cryo vials.
Cell seeding and virus infection:
Seed 1.5 × 106 cells in a T25 cell culture flask and incubate it overnight.
Wash the cell monolayer with 1x DPBS (two times).
Adsorb the monolayer with 2 ml of JEV stock at 0.01 MOI for 90 min at 37°C CO2 incubator.
Wash the cell monolayer with 1x DPBS (two times).
Add 5 ml of DMEM supplemented with 2% FBS and incubate it for 48 hrs at 37°C CO2 incubator.
Virus harvest:
Harvest the cell supernatant after 48 hrs of infection.
Pool the supernatant in sterile tube and centrifuge it at 5000 rpm for 10 min at 4°C.
Pass the supernatant through 0.22 μ syringe filter.
Aliquot the filtered virus suspension in cryovial and store it at −80°C for future use.
3.3 Protocol for the quantitation of JEV by plaque assay
Materials:
Porcine stable kidney cell line (PS cells)
JEV viral stock
Dulbecco’s modified Eagle medium (DMEM, high-glucose for Vero cells) minimum essential medium (MEM for PS cells)
1x DPBS.
24 wells flat bottom plates.
15-ml and 50-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon).
1.5 ml sterile Eppendorf tubes.
2% overlay medium (carboxy methyl cellulose).
Chilled methanol.
1% crystal violet stain.
Cell seeding:
Seed 2 x 105 cells/ well (1 ml) in 10% of MEM.
Incubate it at 37°C CO2 incubator overnight. (Note: during the time of infection, the cells should be 80–90% confluent, forming a monolayer).
Virus infection:
After the incubation, aspirate the old medium from the wells and wash with 1x DPBS.
Prepare a serial 10-fold dilution of JEV in a sterile tube in MEM (without FBS).
Remove the 1x DPBS and add 100 μl of virus dilution to the wells, 100 μl of medium to the cell control well, and incubate it at 37°C CO2 incubator for 90 min.
Swirl the plates every 15–20 min during the incubation time. (During this time, the virus gets adsorbed to the cells.)
Aspirate the virus suspension and wash the wells with 1x DPBS to remove the unabsorbed virus.
Add 1 ml of 2% overlay medium to all the wells starting from cell control.
Incubate the plate for 72 hrs of infection in 37°C CO2 incubator.
Plaque visualization:
Fix the cells with chilled methanol 1 ml/well and keep it for 30 min at −20°C.
Remove methanol and add 1 ml of 1% crystal violet dye for 1 h.
Wash the plate with tap water and observed the plaque.
Calculate the virus titer using the formula.
PFU/ml = (mean foci/well) × (dilution factor)/ (ml inoculum).
3.4 Protocol for the quantitation of JEV using focus forming assay
Materials:
Vero cell line
JEV viral stock
Dulbecco’s modified Eagle medium
1x DPBS.
96 wells tissue culture flat bottom plates.
96-well round bottom plate.
15-ml and 50-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon).
1.5 ml sterile Eppendorf tubes.
2% overlay medium (CMC).
Fixation buffer: 4% paraformaldehyde.
Blocking and permeabilization buffer: 0.1% saponin and 0.1% BSA
Primary antibody: 4G2 monoclonal antibody.
Secondary antibody: horseradish peroxidase (HRP) conjugated anti-mouse IgG.
KPL TrueBlue peroxidase substrate
Rocker 25 (Model S2025-XLD-B).
ELIspot Reader.
Cell seeding:
Using a multichannel pipette, seed 20,000 cells/well in 100 μl of cDMEM in 96 well flat bottom plate.
Incubate at 37°C CO2 incubator for approximately 16–18 hrs or until the cell monolayer reaches ~80% confluency.
Virus infection:
Prepare serial 10-fold dilutions of each sample in a 96-well U-bottom plate using a multichannel pipettor. Mix 20 μl of the sample with 180 μl of DMEM, changing pipet tips between each dilution (Figure 6).
Remove the medium from tissue culture plate and add 100 μl of virus dilution using a multichannel pipette. Discard the tips after dispensing mixture into each row of tissue culture plate.
Incubate the plate at 37°C for 1 hr.
Remove the inoculum from the wells of tissue culture plate. Using a multichannel pipette add 100 μl of warm overlay medium supplemented with 2% FBS.
Incubate it at 37°C CO2 incubator for 24 hrs.
Figure 6.
Schemetic representation and the plate layout for setting up focus forming assay (created with Biorender).
Focus forming assay:
Prepare fixation buffer (4% paraformaldehyde solution) and add 100 μl of this solution onto the overlay medium. (Note: Since the fixation buffer is added onto 100 μl of the infected plate, the final concentration of the fixation buffer becomes 2%).
Incubate at room temperature for 45 min.
Aspirate the overlay medium and the paraformaldehyde using multichannel pipette. Remove any remaining drops by tapping against a stack of paper towels two to three times.
Wash the wells with 150 μl 1x DPBS (two times).
Add 100 μl of the blocking/permeabilization buffer to the wells and incubate it at room temperature for 30 min.
Remove the blocking/permeabilization buffer by inverting the plate and flicking out the buffer. Remove any remaining drops by tapping against a stack of paper towels two to three times.
Dilute 4G2 monoclonal antibody at concentration 1:2000 in blocking/permeabilization buffer. Add 100 μl of the diluted antibody to each well.
Incubate the plate on rocker at room temperature for 2 hrs.
Remove the primary antibody solution and wash the wells with 150 μl of 1X DPBS (2x). Remove any remaining drops by tapping against a stack of paper towels two to three times.
Dilute secondary antibody (horseradish peroxidase (HRP) conjugated anti-mouse IgG) 1:1000 in blocking/permeabilization buffer. Add 100 μl of diluted antibody to each well.
Incubate the plate on rocker at room temperature for 1 hour.
Remove the secondary antibody solution and wash the wells with 150 μl of 1x DPBS (2x). Remove any remaining drops by tapping against a stack of paper towels two to three times.
Add 50 μl of TrueBlue peroxidase substrate to each well.
Incubate it at room temperature on rocker for foci resolution.
Remove the substrate solution and wash the wells with 150 μl of 1x DPBS (2x). Remove any remaining drops by tapping against a stack of paper towels two to three times.
Observe and count the number of foci in ELIspot Reader.
3.5 Protocol for the focus reduction neutralization test (FRNT)
Materials:
Vero cells
JEV viral stock
Human plasma/serum samples.
Dulbecco’s modified Eagle medium (DMEM, high-glucose for Vero cells)
1x DPBS.
96 wells tissue culture flat bottom plates.
Dry heat block/water bath
96 well round bottom plate.
15-ml and 50-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon).
1.5 ml sterile Eppendorf tubes.
2% overlay medium (CMC).
Fixation buffer: 4% paraformaldehyde.
Blocking and permeabilization buffer: 0.1% saponin and 0.1% BSA
Primary antibody: 4G2 monoclonal antibody.
Secondary antibody: horseradish peroxidase (HRP) conjugated anti-mouse IgG.
KPL TrueBlue peroxidase substrate
Rocker 25 (Model S2025-XLD-B).
ELIspot reader.
Cell seeding:
Using a multichannel pipette seed 20,000 cells/well in 100ul of cDMEM in 96 well flat bottom plate.
Incubate at 37°C CO2 incubator for approximately 16–18 hrs or until the cell monolayer reaches ~80% confluency.
Neutralization assay:
Thaw the serum/plasma samples (25-50 μl per tube).
If required, heat inactivate the serum/plasma sample by placing it at 56°C for 30 min.
In 96 well-round bottom plate, aliquot 81 μl of serum-free DMEM into row A. In the remaining rows, add 60 μl of the serum-free DMEM.
Add 9 μl of the plasma/serum samples into row A in duplicate, as shown in the Figure 7.
Mix thoroughly using a multichannel pipette for a minimum of five times.
Transfer 30 μl of samples from row A to row B. With a new set of tips, pipette it up and down three to five times and transfer 30 μl to row C.
Repeat the procedure from row C to row H. Remove 30 μl of the last row H and discard it.
Dilute the JEV strain such that in 60 μl, there are 300–400 FFU.
Add 60 μl of the diluted virus to all the rows using multichannel pipette with a new set of tips for each row and mix thoroughly.
Incubate the plate at 37°C CO2 incubator for 1 hr.
Remove the medium from tissue culture plate with Vero cells and add 100 μl of the virus-serum mixture to the cells using a multichannel pipette. Take a new set of tips for dispensing in each row.
Incubate it at 37°C CO2 incubator for 1 hr.
Remove the virus-serum mixture from the cells and overlay the cells with 100 μl of warm CMC supplemented with 2% FBS.
Incubate the plate at 37°C CO2 incubator for 24 hrs.
Figure 7.
Schemetic representation and plate layout for focus reduction neutralization test (FRNT). Created with Biorender.
Focus forming assay:
Prepare fixation buffer (4% paraformaldehyde solution) and add 100 μl of this solution onto the overlay medium. (Note: Since the fixation buffer is added onto 100 μl of infected plate the final concentration of the fixation buffer becomes 2%).
Incubate at room temperature for 45 min.
Aspirate the overlay medium and the paraformaldehyde using multichannel pipette. Remove any remaining drops by tapping against a stack of paper towels two to three times.
Wash the wells with 150 μl 1x DPBS (2x).
Add 100 μl of the blocking/permeabilization buffer to the wells and incubate it at room temperature for 30 min.
Remove the blocking/permeabilization buffer by inverting the plate and flicking out the buffer. Remove any remaining drops by tapping against a stack of paper towels two to three times.
Dilute 4G2 monoclonal antibody at concentration 1:2000 in blocking/permeabilization buffer. Add 100 μl of the diluted antibody to each well.
Incubate the plate on rocker at room temperature for 2 hrs.
Remove the primary antibody solution and wash the wells with 150 μl of 1x DPBS (2x). Remove any remaining drops by tapping against a stack of paper towels two to three times.
Dilute secondary antibody (Horseradish peroxidase (HRP) Conjugated Anti-Mouse IgG) 1:1000 in blocking/permeabilization buffer. Add 100 μl of diluted antibody to each well.
Incubate the plate on rocker at room temperature for 1 hour.
Remove the secondary antibody solution and wash the wells with 150 μl of 1x DPBS (2x). Remove any remaining drops by tapping against a stack of paper towels two to three times.
Add 50 μl of TrueBlue Peroxidase Substrate to each well of the plate.
Incubate it at room temperature on rocker till observation of the foci.
Remove the substrate solution and wash the wells with 150 μl of 1x DPBS (twice). Remove any remaining drops by tapping against a stack of paper towels two to three times.
The assay plates can be stored at 4°C for up to 1 week, or can be read after the day.
Observe and count the number of foci in ELIspot Reader.
4. Discussion
Plaque assay is a gold-standard method for the detection of infectious virion titer [34, 35]. However, this assay has a few limitations as it is time-consuming, labor-intensive, and has low throughput. Focus forming assay works on the early stages of virus multiplication cycle, where the virus particle is counted within the cells before the cytopathic effect [22]. Moreover, the detection method in focus assay is different and more reproducible than the plaque assay. This makes the focus forming assay a reliable method as it uses a smaller volume of samples and gives the result at a shorter duration as compared to plaque assay. This method can also be used for the detection of neutralizing antibodies against several other arboviruses [23, 24, 25]. The fastest method for the detection of the virus is by using qRT-PCR, but this technique has some limitations as it only confirms the viral genome, and not necessarily the presence of infectious virion. However, if any new assay is developed it should be compared with that of the plaque assay and qRT-PCR for measuring the performance and consistency.
In this study, we identified PS cell line as most appropriate for the plaque assay. It may be due to its superior ability in supporting the productive virus infection than the Vero cells, which were showing tiny plaques in a similar timeframe. However, Vero cells seem to support intracellular virus multiplication in early stages, as observed in the focus forming analyses, where Vero cell line produced a higher titer value as compared to PS cells. Moreover, the extended focus assay at 48 h was not adding any value to the foci resolved in the 24 h assay. While the titers obtained at 24 and 48 hours of infection were the same, the 48-hour termination for JEV revealed significantly larger and fused foci. Thus, these results clearly revealed that the focus forming assay with 24-hour termination is appropriate for the virus quantitation.
Despite the short assay timeframe, the focus forming assay demonstrated comparability to two gold-standard assays—plaque assay and qRT-PCR for virus quantitation. These findings clearly establish the consistency and sensitivity of the focus forming assay. Its applicability was further validated by performing the focus reduction neutralization test (FRNT) to measure virus-neutralizing antibodies in JE patients. The assay successfully quantified neutralizing antibody titers in the patients after 3–4 months of recovery from infection and found that the neutralizing antibody levels remained above the seroprotective threshold. Interestingly, the neutralizing antibody titers were lower against the virus strain of genotype V than the widely circulating genotype III virus. This difference may be attributed to variation in epitopes of the recently emerged genotype V virus, which is more virulent and pathogenic than genotype III. Further studies using the FRNT and other immunological assays are necessary to elucidate the differential immune responses to these two genotypes in the context of infection and vaccination.
Overall, the focus forming assay is a reliable method for detecting the Japanese encephalitis virus and has the potential to replace the traditional gold-standard plaque forming assay. Additionally, the focus reduction neutralization test (FRNT) can be employed to quantify neutralizing antibodies against JEV following vaccination. FRNT is a valuable tool for evaluating the efficacy of JEV vaccines and refining vaccination strategies.
Our study demonstrates that the focus forming assay requires only a small sample volume, operates within a simple and minimal timeframe, and offers the potential for high-throughput applications.
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