Identification, Pathogenicity, and Reverse Genetics System Construction of a Pseudorabies Virus Isolate from Pigs in China
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Virus Strain and Cell Culture
2.3. Isolation of the Virus
2.4. PCR Identification of the Virus
2.5. Phylogenetic Analysis
2.6. Pathogenicity Analysis of PRV-HL-2021 in Mice
2.7. Establishment of a Reverse Genetics Platform for the PRV-HL-2021 Strain
2.8. IFA Test
2.9. Examination of the Replication Kinetics of rPRV-HL-2021
2.10. Plaque Assays
2.11. Generation of a Recombinant PRV Expressing EGFP
2.12. Generation of a Recombinant PRV with Three Gene Knockouts
3. Results
3.1. Isolation and Identification of PRV Epidemic Strain
3.2. The Pathogenicity of PRV-HL-2021
3.3. Construction of a PRV Reverse Genetics Platform Based on the Fosmid Library
3.4. Rescue and Characterization of Recombinant Virus Expressing EGFP
3.5. Rescue of the Recombinant Virus Deleting gI/gE/US9 Genes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No. | Name of the Primer | Sequences of the Primers | Target Genes |
---|---|---|---|
1 | F-identify-gE | TGGCTCTGCGTGCTGTGCTC | gE |
R-identify-gE | CATTCGTCACTTCCGGTTTC | ||
2 | F-identify- gB | GGGGTTGGACAGGAAGGACACCA | gB |
R-identify- gB | AACCAGCTGCACGCGCTCAA | ||
3 | F-US9-rpsL | TCTGCTCGCTGTCCGCGCTACTCGGGGGCATCGTCGCCAGGCACGTGTAGGCTCCCCGCGGGGCTCCTCC | rpsL |
R-US9-rpsL | GGGCGCGGCGGATGGGGGCGGGCCCCCGCTCCCGTTCGCTCGCTCGCTCGCCTCGGCGCCGGCGCACGTC | ||
4 | F-replace rpsL-insert-EGFP | TCTGCTCGCTGTCCGCGCTACTCGGGGGCATCGTCGCCAGGCACGTGTAGTAGTTATTAATAGTAATC | EGFP |
R-replace rpsL-insert-EGFP | GGGCGCGGCGGATGGGGGCGGGCCCCCGCTCCCGTTCGCTCGCTCGCTCGGCAGTGAAAAAAATGCTT | ||
5 | F-delgE/gI/US9-rpsL | GCCTCCGCAGTACCGGCGTCGATGATGATGGTGGCGCGCGACGTGACCCGGCAGTGAAAAAAATGCTT | rpsL |
R-delgE/gI/US9-rpsL | TCTAGGAGATGGTACATCGCGGGGCGCGCTCGCGTCCGTTGCCGCGCCCGTCAGAAGAACTCGTCAAGAAGGCG | ||
6 | F-replace rpsL-delgE/gI/US9 | TCTAGGAGATGGTACATCGCGGGGCGCGCTCGCGTCCGTTGCCGCGCCCGCCTCGGCGCCGGCGCACGTC | Parts of US2 and US7 |
PRV Strain | Country | Year of Isolating | GenBank No. |
---|---|---|---|
HNX | China | 2012 | KM189912.1 |
HuBXY/2018 | China | 2018 | MT468549.1 |
HN1201 | China | 2012 | KP722022.1 |
HNB | China | 2015 | KM189914.1 |
hSD-1/2019 | China | 2019 | MT468550.1 |
TJ | China | 2014 | KJ789182.1 |
SD18 | China | 2020 | MT949536.1 |
PRV-GD | China | 2021 | OK338076.1 |
HLJ8 | China | 2013 | KT824771.1 |
HeN1 | China | 2012 | KP098534.1 |
PRV-JM | China | 2021 | OK338077.1 |
JX/CH/2016 | China | 2016 | MK806387.1 |
JS-2012 | China | 2012 | KP257591.1 |
Fa | China | 1980s | KM189913.1 |
Ea | China | 1990s | KU315430.1 |
SC | China | 1986 | KT809429.1 |
Becker | USA | 1979 | JF797219.1 |
Bartha | Hungary | 1961 | JF797217.1 |
Kolchis | Greece | 2010 | KT983811.1 |
Kaplan | USA | 1959 | JF797218.1 |
The Number of Animals That Can Detect PRV | ||||||||
---|---|---|---|---|---|---|---|---|
Strain | Dose (TICD50) | Route | Brain | Heart | Liver | Spleen | Lung | Kidney |
PRV-HL-2021 | 105 | i.p. | 4/5 | 1/5 | 0/5 | 0/5 | 0/5 | 0/5 |
104 | i.p. | 2/5 | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | |
103 | i.p. | 2/5 | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | |
102 | i.p. | 2/5 | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | |
DMEM | 0.1 mL | i.p. | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 |
Fosmid | Relative Position in the Genome (nt) | The Size of the Fragment (bp) | Fosmid | Relative Position in the Genome (nt) | The Size of the Fragment (bp) |
---|---|---|---|---|---|
PRV-IF-a | 1–44102 | 44,101 | PRV-IF-g | 50226–89728 | 39,502 |
PRV-IF-b | 1–39716 | 39,515 | PRV-IF-h | 51898–85801 | 33,903 |
PRV-IF-c | 33891–68519 | 34,628 | PRV-IF-i | 67889–102713 | 34,824 |
PRV-IF-d | 28749–61624 | 32,875 | PRV-IF-j | 91337–122601 | 25,883 |
PRV-IF-e | 47680–10987 | 35,793 | PRV-IF-k | 100865–end | 26,640 |
PRV-IF-f | 61229–101922 | 40,693 | PRV-IF-l | 101794–end | 25,711 |
Group | Fosmid Combination | CPE Occurrence Time (hpt) |
---|---|---|
1 | a + c + e + j + k | no CPE occurrence |
2 | a + c + f + j + k | 24 |
3 | b + d + f + g + h + i | 48 |
4 | b + d + h + i + k | 36 |
5 | b + d + h + i + j + k | 54 |
6 | a + c + e + j + l | no CPE occurrence |
7 | a + c + f + j + l | 36 |
8 | b + d + f + g + h + l | 54 |
9 | b + d + h + i + l | 36 |
10 | b + d + h + i + j + l | 36 |
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Zhou, M.; Liang, H.; Nie, N.; Zhang, L.; Zhu, R.; Cao, S.; Zhu, S. Identification, Pathogenicity, and Reverse Genetics System Construction of a Pseudorabies Virus Isolate from Pigs in China. Vet. Sci. 2025, 12, 519. https://doi.org/10.3390/vetsci12060519
Zhou M, Liang H, Nie N, Zhang L, Zhu R, Cao S, Zhu S. Identification, Pathogenicity, and Reverse Genetics System Construction of a Pseudorabies Virus Isolate from Pigs in China. Veterinary Sciences. 2025; 12(6):519. https://doi.org/10.3390/vetsci12060519
Chicago/Turabian StyleZhou, Mo, Haiyang Liang, Nannan Nie, Li Zhang, Rui Zhu, Shinuo Cao, and Shanyuan Zhu. 2025. "Identification, Pathogenicity, and Reverse Genetics System Construction of a Pseudorabies Virus Isolate from Pigs in China" Veterinary Sciences 12, no. 6: 519. https://doi.org/10.3390/vetsci12060519
APA StyleZhou, M., Liang, H., Nie, N., Zhang, L., Zhu, R., Cao, S., & Zhu, S. (2025). Identification, Pathogenicity, and Reverse Genetics System Construction of a Pseudorabies Virus Isolate from Pigs in China. Veterinary Sciences, 12(6), 519. https://doi.org/10.3390/vetsci12060519