Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Antibody
2.2. Clinical Samples Collection
2.3. Virus Isolation and Propagation
2.4. Growth Kinetics and Virus Titration
2.5. Immunofluorescence Assay (IFA)
2.6. Morphologic Observation of Virus by TEM
2.7. RNA Extraction, RT-PCR and Sequence Analysis
2.8. Pig Infection Experiment
2.9. Detection of Cytokines
2.10. Statistical Analysis
3. Results
3.1. Virus Isolation and In Vitro Characterization
3.2. Morphologic Characterization of Virus
3.3. Sequence Analysis
3.4. Pathogenicity of SDSX16
3.5. Analysis of Cytokine Expression by SDSX16 Infected Porcine Intestinal Epithelial Cells
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence 5′–3′ | Size (bp) |
---|---|---|
PEDV-F1 | CGCAAATGGGCGGTAGGCGTG | 1169 |
PEDV-R1 | GCATGAAGCCACAAGGAC | |
PEDV-F2 | GTTGTCTTGTCTGAGCCA | 1119 |
PEDV-R2 | CAGGATGTTCAATGACAA | |
PEDV-F3 | CAATGTGAATCTCGTCGT | 1259 |
PEDV-R3 | GATCCTGTGAGATCGTGTC | |
PEDV-F4 | CTCCCTTGAATTTGAGTTCG | 1326 |
PEDV-R4 | TGACACCACGTCCAACTTTA | |
PEDV-F5 | CAGACGTTGAGCCTGTATT | 1157 |
PEDV-R5 | GTTGTCTGTGACAACGGT | |
PEDV-F6 | ATGTGGAGCGTTTCTACGCA | 1284 |
PEDV-R6 | AACATCATCACAAACAGGGC | |
PEDV-F7 | GCCTTTCGTAAGAGGGAT | 1460 |
PEDV-R7 | AGGGCAATGAAATCACGTA | |
PEDV-F8 | TTGCTGAGGCTCATCGTTA | 1493 |
PEDV-R8 | AGAGCCTACGAACTTGTCA | |
PEDV-F9 | TCGGTGATATGTCTGTTGGC | 1346 |
PEDV-R9 | TGCAGAGACTGGATTGAGGC | |
PEDV-F10 | TGGTACCGTTGAGTTTTGCT | 1330 |
PEDV-R10 | TTCATGCTAGAGAGACAGCC | |
PEDV-F11 | GCTTCTACGGTATTCTCTACT | 1249 |
PEDV-R11 | TGCACCATTAGGAGAATCCA | |
PEDV-F12 | CCTGGTAAGCTGAAGCAGCG | 1462 |
PEDV-R12 | AAGCACTCACTAGCAAGGCA | |
PEDV-F13 | ATTCATCGTGTCTATGCATTG | 1367 |
PEDV-R13 | TGCTCTGTCGCACTTTGGG | |
PEDV-F14 | CCATGACTACTCGGCAGTAT | 1232 |
PEDV-R14 | CACACATATGGAGTGATGGC | |
PEDV-F15 | AATCTGCAGGGCTTTGTGTT | 1318 |
PEDV-R15 | GACAGCGATAACACTTGTGC | |
PEDV-F16 | TGGTGGATGAGGTCTCTATG | 1342 |
PEDV-R16 | GGGCATGTTTGAAACAACAG | |
PEDV-F17 | GGTTGCGTTGTAACTGAGTC | 1358 |
PEDV-R17 | GTCCTACCTTACGTTTGGCA | |
PEDV-F18 | TCGGCGGGCTTTACTATTTG | 1306 |
PEDV-R18 | CACCAAGATGTAGCACACG | |
PEDV-F19 | CTCCAGACATTTTATCCGCA | 1294 |
PEDV-R19 | CGCGCAGTAGCATTAGTGTT | |
PEDV-F20 | GGCGGTTATCTACCTATTGG | 1345 |
PEDV-R20 | CTGTTCATGACTCAGAAGG | |
PEDV-F21 | TGTTGGACGCTGTCACAAT | 1518 |
PEDV-R21 | GACCATTAGAACAGCGCTTA | |
PEDV-F22 | TCACCCAGTACACTGCAGC | 1412 |
PEDV-R22 | GAGAGACTCTGAACGCTGC | |
PEDV-F23 | GCCTTGACTCTACGTGAGCC | 1423 |
PEDV-R23 | CGCTATTACACAACCGGTGA | |
PEDV-F24 | GACACTTTCTTTCCTCAATG | 1475 |
PEDV-R24 | AGTCCTATAGCGGAGGTCGG | |
PEDV-F25 | GGACGTGTTGGTCGTTCAGT | 1242 |
PEDV-R25 | CATTTGGATCAGACTTTGGC | |
PEDV-F26 | AGATGCGGAATTTGTCGAA | 1121 |
PEDV-R26 | GACTTCAAGTCACGTGAA | |
5′UTR1 | GCATCGCTCCATTATCC | 623 |
5′UTR2 | TTACCACCACGACGACC | |
3′UTR1 | GGCATAAGCAACAGCAG | 690 |
3′UTR2 | ATCGCCAGTTTAGCACCA |
Gene | Sequence 5′–3′ | GenBank NO. |
---|---|---|
IL-1β-F | GAAAGCCATACCCAGAGGTC | NM_214055.1 |
IL-1β-R | GCACTAATCTAGGGAAGACAGC | |
IL-6-F | TGGGTTCAATCAGGAGACCT | AF518322.1 |
IL-6-R | CAGCCTCGACATTTCCCTTA | |
TNF-α-F | CCCCCAGAAGGAAGAGTTTC | JF831365.1 |
TNF-α-R | CGGGCTTATCTGAGGTTTGA | |
IFN-β-F | CTCCAAATCGCTCTCCTGAT | GQ415073.1 |
IFN-β-R | GGGACCTCAAAGTTCATCCT | |
β-actin-F | TCTGGCACCACACCTTCT | AY550069.1 |
β-actin-R | GATCTGGGTCATCTTCTCAC |
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Zhao, P.; Wang, S.; Chen, Z.; Yu, J.; Tang, R.; Qiu, W.; Zhao, L.; Liu, Y.; Guo, X.; He, H.; et al. Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus. Viruses 2020, 12, 391. https://doi.org/10.3390/v12040391
Zhao P, Wang S, Chen Z, Yu J, Tang R, Qiu W, Zhao L, Liu Y, Guo X, He H, et al. Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus. Viruses. 2020; 12(4):391. https://doi.org/10.3390/v12040391
Chicago/Turabian StyleZhao, Pengwei, Song Wang, Zhi Chen, Jiang Yu, Rongzhi Tang, Wenbin Qiu, Lu Zhao, Yueyue Liu, Xiaozhen Guo, Hongbin He, and et al. 2020. "Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus" Viruses 12, no. 4: 391. https://doi.org/10.3390/v12040391