Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells, Viruses and Plasmids
2.2. Virus Isolation and Purification
2.3. Neutralizing Test
2.4. PCR Amplification of Full-Length gB, gE, gC, gD, TK, RR1 and RR2 of HNXY and Phylogenetic Analysis
2.5. One-Step Growth Analysis
2.6. Construction of Transfer Vectors
2.7. Generation of rHNXY-∆TK/∆gE
2.8. Animal Experiment
2.8.1. Experimental Infection of Mice
2.8.2. Experimental Infection of Pigs
2.9. Accession Numbers
2.10. Statistical Analyses
3. Results
3.1. Virus Isolation
3.2. Genetic Variation Analysis of gB, gE, gC, gD, TK, RR1 and RR2 Proteins
3.3. Phylogenetic Tree Analysis
3.4. Generation of rHNXY-∆TK/∆gE
3.5. Growth Properties of rHNXY-∆TK/∆gE and HNXY In Vitro
3.6. Pathogenicity of rHNXY-∆TK/∆gE and HNXY In Vivo
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence | Details |
---|---|---|
gE-U-F | 5-ccggaattccctcctcgccgccctgaccctg-3 | upstream homologous fragments flanking gE gene |
gE-U-R | 5-ggactagtgacggagataaaacgccaccca-3 | |
gE-D-F | 5-ggactagtataccgggagaaccggt-3 | downstream homologous fragments flanking gE gene |
gE-D-R | 5-cccaagcttaggagcggttgtgga-3 | |
TK-U-F | 5-ccggaattcccggttgcccacgtacag-3 | upstream homologous fragments flanking TK gene |
TK-U-R | 5-ggactagtcccggcgcgcttccgg-3 | |
TK-D-F | 5-ggactagtccctcgcccctcccaccc-3 | downstream homologous fragments flanking TK gene |
TK-D-R | 5-cccaagcttccgggtcctcgccgaa-3 | |
loxP-EGFP-F | 5-ggactagtataacttcgtatagcatacattatacgaagttattagttattaatagta-3 | EGFP expression cassette |
loxP-EGFP-R | 5-ggactagtataacttcgtataatgtatgctatacgaagttatagccatagagcccac-3 | |
gE-U-L | 5-agcccggtccgtagcctccgcagtac-3 | gE deletion verification |
gE-D-R | 5-cctccgtccactcgccggcgt-3 | |
TK-U-L | 5-ggtgcaccaggtgcaggcacag-3 | TK deletion verification |
TK-D-R | 5-ggcgacggtcgcccgcgcgagg-3 |
Gene | Compared with Bartha, Kaplan and Becker | Compared with SC, LA, Ea, Fa | Compared with PRV Isolated after 2012 in China | |||
---|---|---|---|---|---|---|
Nucleotide Sequence Homology | Amino Acid Sequence Homology | Nucleotide Sequence Homology | Amino Acid Sequence Homology | Nucleotide Sequence Homology | Amino Acid Sequence Homology | |
gB | 98.2%–98.4% | 96.4%–97.2% | 99.5%–99.6% | 98.7%–98.9% | 99.7%–99.8% | 99.3%–99.9% |
gC | 95.9%–96.2% | 91.7%–92.5% | 96.9%–99.9% | 94.0%–99.4% | 99.5%–99.6% | 98.6%–98.8% |
gD | 98.5%–98.8% | 96.0%–97.5% | 99.2%–99.4% | 98.3%–98.5% | 99.4%–99.6% | 98.3%–98.5% |
gE | 97.6%–97.8% | 95.5%–95.7% | 99.4%–99.5% | 98.8%–99.3% | 99.7%–99.9% | 99.0%–99.7% |
TK | 99.6%–99.7% | 99.1%–99.4% | 99.7%–100% | 99.1%–99.7% | 99.9%–100% | 99.4%–99.7% |
RR1 | 98.6% | 96.8%–97.1% | 99.7%–99.9% | 99.2%–99.5% | 99.7%–99.9% | 99.2%–99.5% |
RR2 | 98.4%–99.5% | 97.4%–99.0% | 99.5%–99.9% | 99.0%–99.3% | 99.7%–99.9% | 99.0%–99.3% |
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Wu, F.; Lv, Y.; Zhang, S.; Liu, L.; Wu, Y.; Zhao, P.; Peng, Z.; Liu, S.; Zhang, Z.; Li, W. Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE. Animals 2020, 10, 1804. https://doi.org/10.3390/ani10101804
Wu F, Lv Y, Zhang S, Liu L, Wu Y, Zhao P, Peng Z, Liu S, Zhang Z, Li W. Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE. Animals. 2020; 10(10):1804. https://doi.org/10.3390/ani10101804
Chicago/Turabian StyleWu, Fengsun, Yujin Lv, Shijun Zhang, Lingling Liu, Yuchen Wu, Pandeng Zhao, Zhifeng Peng, Shengli Liu, Zhonghua Zhang, and Wengang Li. 2020. "Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE" Animals 10, no. 10: 1804. https://doi.org/10.3390/ani10101804