The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments
Abstract
:1. Introduction
2. Results
2.1. Experimental Design
2.2. The Viral Loads and Titers of BTV2/BTV12 in Infected PBMCs
2.3. Infection Rate of BTV2 and BTV12 in MDMs Detected by IFA
2.4. Expression of mRNA in BTV-Infected PBMCs
2.5. The Cytokine Production Induced by BTV2 and BTV12
2.6. Prediction of Potential IL-4-Inducing Peptides on the VP2 of Various BTV Strains
2.7. Predicted IL-4-Inducing Peptide Segments in the VP2 Tip Region of BTV2 and BTV12
2.8. Induction of IL-4 Production in PBMC Stimulated with Synthetic Peptide
3. Discussion
4. Materials and Methods
4.1. Animals, and Preparation of Bovine PBMC and Monocyte-Derived Macrophage (MDMs)
4.2. Virus Infection and Quantitation of BTV Viral Loads in PBMCs
4.3. TCID50 Assay to Determine the Titer of Progeny Virus in the PBMC Supernatant
4.4. Pair-Wise Comparison of the Cytokine mRNA Expression by qRT-PCR
4.5. IFA of BTV-Infected MDMs by Confocal Microscopy
4.6. Enzyme-Linked Immunosorbent Assay (ELISA) for IL-1β, IFN-α, and IL-4
4.7. The Prediction of Potential IL-4-Inducing Peptides and Three-Dimensional (3D) Protein Structure Modeling on VP2 of BTVs
4.8. Synthesis of BTV Potential IL-4 Inducing Peptides
4.9. PBMC Stimulation Assay for Synthetic IL-4-Inducing Peptides
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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BTV Serotype | Allergy-Reported | Non-Allergy Reported | |||
---|---|---|---|---|---|
Number | 2, 8, 11, 17 (n = 20) | 12, 1 (n = 15) | 10 (n = 7) | p Value (One-Way ANOVA) | |
Tip region a | 7.20 ± 0.748 | 2.60 ± 0.800 | 4.286 ± 0.452 | <0.01 | |
Total VP2 b | 24.80 ± 2.713 | 19.80 ± 2.926 | 21.714 ± 1.666 | <0.01 | |
Tip/Total c | 0.290 ± 0.0469 | 0.131 ± 0.0462 | 0.197 ± 0.0104 | <0.01 |
Parameters | BTV2 | BTV12 |
---|---|---|
Replication (VP7) in bovine PBMC (Figure 2) | Parallel | Parallel, slightly higher |
Replication (TCID50) in bovine PBMC (Figure 2) | Parallel | Parallel, more virulent |
Infection rates in MDMs (Figure 3) | 75% at MOI of 1 at 12 hpi | Cells died and detached at MOI of 1 by 12 hpi; 76.1% at MOI of 0.5. Stronger affinity or virulence to MDM/PBMC |
Innate immunity (TNF-α and IL-1β) (Figure 4, Figure 5 and Figure 8) | Minimal to mild | Activated strong and sustained, 2–4-fold higher (TNF-α) at 6–12 hpi 17–27-fold higher (IL-1β) at 6–12 hpi Likely part of a cytokine storm, which is cytocidal (combined Figure 3 with Figure 4c) |
Acquired immunity (Th2 and IL-4) (Figure 4, Figure 5 and Figure 8) | Activated strong and sustained 6–11-fold higher | Minimal |
Number of potential IL-4-inducing segments in the tip region of VP2 (Figure 6) | 7 | 2 |
Association with allergy (Table 1, Supplement Tables S1 and S2) | Yes | Low |
IL-4-inducing capacity of synthetic peptides (Figure 7) | Yes | Low |
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Yang, J.-L.; Chang, C.-Y.; Sheng, C.-S.; Wang, C.-C.; Wang, F.-I. The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments. Pathogens 2021, 10, 3. https://doi.org/10.3390/pathogens10010003
Yang J-L, Chang C-Y, Sheng C-S, Wang C-C, Wang F-I. The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments. Pathogens. 2021; 10(1):3. https://doi.org/10.3390/pathogens10010003
Chicago/Turabian StyleYang, Jia-Ling, Chia-Yi Chang, Chih-Shuan Sheng, Chia-Chi Wang, and Fun-In Wang. 2021. "The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments" Pathogens 10, no. 1: 3. https://doi.org/10.3390/pathogens10010003