Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells
2.2. Isolation of PED Virus
2.3. Live Attenuated PEDV
2.4. Titration of PEDV
2.5. Immunofluorescence Assay
2.6. Evaluation of Pathogenicity
2.7. Quantitative Real-Time RT-PCR
2.8. Histopathology and Immunochemistry
2.9. Next-Generation Sequencing (NGS)
2.10. Phylogenetic Tree
3. Results
3.1. Isolation of PEDV from Porcine Intestines
3.2. Development of Live Attenuated Vaccine Candidates
3.3. Growth Characteristics of CKT-7 Strains
3.4. Growth Kinetics of CKT-7 Strains
3.5. Evaluation for Pathogenicity of CKT-7 Strains
3.6. Analysis of Histopathology and Immunohistochemistry (IHC)
3.7. Analysis of the Phylogenetic Relationship
3.8. Comparison of Whole-Genome Sequence
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains | Passage Number | ||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Origin | 1 | 2 | 3 | …**** | 9 | 10 | 11 | … | 32 | 33 | 34 | … | 39 | 40 | 41 | 42 | … | 51 | 52 | 53 | 54 | … | 60 | 61 | 62 | 63 | 64 | 65 | 66 | … | 70 | 71 | 72 | … | 180 |
T15 * | 1 | 2 | 3 | … | 9 | 10 | 11 | … | 32 | 33 | 34 | … | 39 | 40 | 41 | 42 | … | 51 | 52 | 53 | 54 | … | 60 | 61 | 62 | 63 | 64 | 65 | 66 | … | 70 | 71 | 72 | … | 180 |
T15N ** | 1 | 2 | 3 | … | 24 | 25 | 26 | … | 31 | 32 | 33 | 34 | … | 43 | 44 | 45 | 46 | … | 52 | 53 | 54 | 55 | 56 | 57 | 58 | … | 62 | 63 | 64 | … | 172 | ||||
T10N | 1 | 2 | 3 | … | 8 | ||||||||||||||||||||||||||||||
T5N | 1 | 2 | 3 | … | 12 | ||||||||||||||||||||||||||||||
T2N | 1 | 2 | 3 | … | 9 | ||||||||||||||||||||||||||||||
N | 1 | 2 | 3 | 4 | 5 | 6 | … | 10 | 11 | 12 | … | 120 | |||||||||||||||||||||||
NF *** | 1 | 2 | 3 | … | 7 | 8 | 9 | … | 117 | ||||||||||||||||||||||||||
X | 1 | 2 | 3 | … | 111 | ||||||||||||||||||||||||||||||
F | 1 | 2 | … | 110 |
T15 | T15N | N | NF | F | X | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Passage No. | Titer * | Passage No. | Titer | Passage No. | Titer | Passage No. | Titer | Passage No. | Titer | Passage No. | Titer |
P10 | 3.50 ± 0.00 | P10 | 3.67 ± 0.14 | P10 | P10 | P10 | P10 | ||||
P20 | 4.58 ± 0.14 | P20 | 4.75 ± 0.00 | P20 | P20 | P20 | P20 | ||||
P30 | 5.42 ± 0.29 | P30 | 5.92 ± 0.14 | P30 | P30 | P30 | P30 | ||||
P40 | 6.00 ± 0.25 | P40 | 5.42 ± 0.14 | P40 | P40 | P40 | P40 | ||||
P50 | 6.17 ± 0.14 | P50 | 6.50 ± 0.25 | P50 | P50 | P50 | P50 | ||||
P60 | 5.33 ± 0.29 | P60 | 5.33 ± 0.52 | P60 | P60 | P60 | P60 | ||||
P70 | 6.08 ± 0.38 | P70 | 5.50 ± 0.00 | P70 | P70 | P70 | P70 | ||||
P80 | 6.00 ± 0.00 | P80 | 5.85 ± 0.14 | P80 | 7.17 ± 0.14 | P80 | 6.67 ± 0.14 | P80 | 6.33 ± 0.14 | P80 | 6.75 ± 0.00 |
P90 | 6.33 ± 0.14 | P90 | 5.75 ± 0.43 | P90 | 7.08 ± 0.29 | P90 | 7.00 ± 0.43 | P90 | 6.83 ± 0.38 | P90 | 6.92 ± 0.38 |
P100 | 5.50 ± 0.00 | P100 | 4.83 ± 0.14 | P100 | 7.25 ± 0.43 | P100 | 5.92 ± 0.14 | P100 | 6.83 ± 0.14 | P100 | 6.75 ± 0.25 |
P110 | 6.58 ± 0.14 | P110 | 7.08 ± 0.14 | P110 | 7.42 ± 0.38 | P110 | 6.67 ± 0.52 | P110 | 6.75 ± 0.00 | P110 | 6.42 ± 0.29 |
P120 | 6.33 ± 0.14 | P120 | 6.92 ± 0.38 | P120 | 7.83 ± 0.29 | P120 | 7.83 ± 0.29 | P120 | 7.00 ± 0.00 | P120 | 7.17 ± 0.52 |
P130 | 6.58 ± 0.14 | P130 | 6.42 ± 0.29 | P130 | 8.25 ± 0.25 | P130 | 7.08 ± 0.38 | P130 | 7.08 ± 0.29 | P130 | 6.83 ± 0.38 |
P140 | 7.42 ± 0.14 | P140 | 7.67 ± 0.29 | P140 | 8.67 ± 0.29 | P140 | 7.28 ± 0.29 | P140 | 7.33 ± 0.14 | P140 | 7.25 ± 0.25 |
P150 | 8.30 ± 0.14 | P150 | 8.50 ± 0.00 | P150 | 8.50 ± 0.25 | P150 | 7.08 ± 0.14 | P150 | 7.17 ± 0.14 | P150 | 7.42 ± 0.52 |
P160 | 7.33 ± 0.38 | P160 | 7.08 ± 0.29 | P160 | 8.25 ± 0.66 | P160 | 8.33 ± 0.14 | P160 | 7.92 ± 0.14 | P160 | 7.83 ± 0.14 |
P170 | 7.25 ± 0.25 | P170 | 7.50 ± 0.25 | P170 | 8.00 ± 0.00 | P170 | 7.67 ± 0.14 | P170 | 7.00 ± 0.25 | P170 | 6.83 ± 0.29 |
P180 | 7.00 ± 0.00 | P180 | 7.83 ± 0.14 | P180 | 8.25 ± 0.25 | P180 | 7.92 ± 0.14 | P180 | 7.25 ± 0.25 | P180 | 6.92 ± 0.14 |
Group | Inoculum | Route | No. of Pigs | Mortality Rate * [% (No/Total)] | Severe Diarrhea Rate [% (No/Total)] | Clinical Symptoms | Virus Shedding | Peak Fecal Virus Shedding Titer [log10 Copies/μL], dpi |
---|---|---|---|---|---|---|---|---|
1 | Positive control | Oral | 3 | 100 (3/3) | 100 (3/3) | NA ** (>4) | Started at 1 dpi | 5.05 ± 0.24, 2 |
2 | Negative control | 3 | 0 (0/3) | 0 (0/3) | No diarrhea | N/D *** | NA | |
3 | CKT-7 T15 | 3 | 0 (0/3) | 0 (0/3) | Mild | Limited | NA | |
4 | CKT-7 T15N | 3 | 0 (0/3) | 0 (0/3) | Mild | Started at 4 dpi | 2.48 ± 0.13, 5 | |
5 | CKT-7 N | 3 | 0 (0/3) | 0 (0/3) | No diarrhea | N/D | NA | |
6 | CKT-7 NF | 3 | 0 (0/3) | 0 (0/3) | Mild | Limited | NA | |
7 | CKT-7 F | 3 | 0 (0/3) | 0 (0/3) | No diarrhea | N/D | NA | |
8 | CKT-7 X | 3 | 0 (0/3) | 0 (0/3) | No diarrhea | Started at 2 dpi | 2.68 ± 0.49, 3 |
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Kim, D.-M.; Moon, S.-H.; Kim, S.-C.; Cho, H.-S.; Tark, D. Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity. Vaccines 2023, 11, 923. https://doi.org/10.3390/vaccines11050923
Kim D-M, Moon S-H, Kim S-C, Cho H-S, Tark D. Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity. Vaccines. 2023; 11(5):923. https://doi.org/10.3390/vaccines11050923
Chicago/Turabian StyleKim, Dae-Min, Sung-Hyun Moon, Seung-Chai Kim, Ho-Seong Cho, and Dongseob Tark. 2023. "Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity" Vaccines 11, no. 5: 923. https://doi.org/10.3390/vaccines11050923
APA StyleKim, D.-M., Moon, S.-H., Kim, S.-C., Cho, H.-S., & Tark, D. (2023). Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity. Vaccines, 11(5), 923. https://doi.org/10.3390/vaccines11050923