Characterization of BoHV-1 gG-/tk-/gE- Mutant in Differential Protein Expression, Virulence, and Immunity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Cell Culture and Virus Strains
2.3. Construction of Triple Mutant Virus BoHV-1 Strain gG-/tk-/gE-
2.3.1. Construction of Transfer Vector pBoHV-1 gE-
2.3.2. Generation and Characterization of the Triple Mutant Virus
2.4. Viral Growth Kinetics and Plaque Size Determination
2.5. Label Free Quantitative Proteomics (LFQP)
2.5.1. Virions Purification
2.5.2. LC-MS/MS Analysis
2.5.3. Database Search and Bioinformatic Analysis
2.6. Cattle Experiments
2.6.1. Clinical Evaluation
2.6.2. Virus Isolation and Titration
2.6.3. Serological Investigation
2.6.4. Histo-Pathological Examination of Lungs
2.7. Statistical Analysis
3. Results
3.1. Construction and Characterization of the Recombinant BoHV-1 gG-/tk-/gE- Triple- Deleted Mutant
3.2. Comparative Viral Growth Kinetics and Plaque Size Estimation
3.3. Comparative Proteomics of the Three BoHV-1 Strains
3.3.1. Differentially Expressed Proteins in Different Comparable Strains
3.3.2. GO Secondary Annotation Classification
3.4. In Vivo Experiment Findings
3.4.1. Virulence and Reactivation of BoHV-1 gG-/tk-/gE- in Calves
3.4.2. Protection of BoHV-1 gG-/tk-/gE- against wt BoHV-1 Challenge in Calves
3.4.3. Cross-Protection of BoHV-1 gG-/tk-/gE- against wt BoHV-5 Challenge in Calves
3.5. Serological Investigations
3.5.1. Humoral Immune Response in Calves
3.5.2. Cytokine Production in Calves
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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Animal | Virus | No | Vaccinated Route and Dose | Dex Injection a | Challenge Virus | Challenge Date and Dose | Euthanasia and Necropsy |
---|---|---|---|---|---|---|---|
Calf | BoHV-1 gG-/tk-/gE- | 6 | IN, 4 × 107 PFU | 21–25 dpi | wt BoHV-1 (3) b wt BoHV-5 (3) b | 35 dpi, 4 × 107 PFU | 28 dpc |
BoHV-1 gG-/tk- | 6 | IN, 4 × 107 PFU | 21–25 dpi | wt BoHV-1 (3) b wt BoHV-5 (3) b | 35 dpi, 4 × 107 PFU | 28 dpc | |
wt BoHV-1 | 3 | IN, 4 × 107 PFU | 21–25 dpi | 35 dpi, 4 × 107 PFU | 28 dpc | ||
Unvaccinated control | 6 | DMEM | 21–25 dpi | wt BoHV-1 (3) b wt BoHV-5 (3) b | 35 dpi, 4×107 PFU | 28 dpc | |
Negative control | 3 | 28 dpc |
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Marawan, M.A.; Deng, M.; Wang, C.; Chen, Y.; Hu, C.; Chen, J.; Chen, X.; Chen, H.; Guo, A. Characterization of BoHV-1 gG-/tk-/gE- Mutant in Differential Protein Expression, Virulence, and Immunity. Vet. Sci. 2021, 8, 253. https://doi.org/10.3390/vetsci8110253
Marawan MA, Deng M, Wang C, Chen Y, Hu C, Chen J, Chen X, Chen H, Guo A. Characterization of BoHV-1 gG-/tk-/gE- Mutant in Differential Protein Expression, Virulence, and Immunity. Veterinary Sciences. 2021; 8(11):253. https://doi.org/10.3390/vetsci8110253
Chicago/Turabian StyleMarawan, Marawan A., Mingliang Deng, Chen Wang, Yingyu Chen, Changmin Hu, Jianguo Chen, Xi Chen, Huanchun Chen, and Aizhen Guo. 2021. "Characterization of BoHV-1 gG-/tk-/gE- Mutant in Differential Protein Expression, Virulence, and Immunity" Veterinary Sciences 8, no. 11: 253. https://doi.org/10.3390/vetsci8110253