Characteristics and vaccine potential of genetically engineered replication-competent and replication-incompetent viral hemorrhagic septicemia viruses (VHSVs)
- Alternative Title
- 분열가능 및 분열불능 다양한 재조합 VHSV 제작, 특성 분석 및 백신 효과
- Abstract
- everse genetics technology can be used as a tool for developing live attenuated vaccines against specific viruses and delivery of foreign genes as well as identifying genes functions by observing the phenotype changes of cells and genetically engineered viruses. In this research, we conducted several studies on live attenuated vaccines that can effectively induce immune responses by confirming the compensatory mutation characteristics caused by artificial mutation of the VHSV M gene using based on the recombinant VHSVs.
The M gene of VHSV is known to control viral transcription and viral genome replication, in addition to diverse immunological responses of host cells. In this study, an artificially mutant recombinant virus was rescued by substitution at codon 62 and codon 181 to alanine in the matrix gene. However, during the course of recombinant VHSV rescue, secondary compensatory mutation in M gene was confirmed. To verify characteristics of compensatory mutation by artificial mutation, M gene-mutated rVHSVs contain D62A, E181A or D62AE181A in M gene were rescued several times, then viruses from passage P1 to P3 were collected and sequenced. As a result of sequence in M gene of rescued M gene-mutated rVHSVs, we figured out that the codon 181 has an important effect on compensatory mutation. In addition, virus replication and host immune response were confirmed from M gene-mutated rVHSVs and rVHSVs haboring secondary mutation, and as a results, it was confirmed that most of the compensatory mutation in viruses has deleterious effect of the engineered mutation. Therefore, the reversion to virulent form through compensatory mutations caused by artificial mutations in VHSV suggests that live attenuated virus-based vaccines, rescued based on point mutation, are unsafe to be used as vaccines.
Interferon is a secreted protein (cytokine) that induces an antiviral state in cells and plays an important role in defense mechanism against viral infection. To investigate the function of newly reported olive flounder interferon 4 (OFIFN4),eGFP- and OFIFN4-expressing EPC cell lines were established. OFIFN4-expressing EPC cells have a protective effect against VHSV. Based on this result, OFIFN4-expressing rVHSVs were rescued and confirmed as a potential vaccine candidate against VHSV.
C-type lectin (CTL) is a molecule that lead to innate immune response by recognizing carbohydrates on the surface of pathogens. CTLs have a number of motifs that can bind to specific sugars, and since the specificity of sugars is determined by those motifs, so they play a very important role in CTLs. In this study, CTL-expressing rVHSVs were produced, and stimulated innate immune response was confirmed by respiratory burst activity in olive flounder’s phagocytes.
The results of previous studies confirmed that the live-attenuated vaccine has effectively induced the immune response. However, there are still difficulties in commercial use due to safety concerns. In this study, various single-cycle live viruses were rescued to overcome the safety of live attenuated virus-based vaccines. Single-cycle live virus is a very safe form of live vaccine type because it cannot replicate normally due to genetically defective essential genes, and can only replicate in cells that supply those genes.
Interferon-γ (IFNγ) is classified as a Type II interferon and plays an important role in innate and adaptive immunity against infectious pathogens. Based on previous rescued rVHSV-A-IFNγ, we rescued a single-cycle rVHSV (rVHSV-A-IFNγ-ΔG) that lacks the G gene and contains olive flounder IFNγ and evaluated its potential protection through the immunization of olive flounder. In comparison with rVHSV-ΔG, we confirmed a protective efficacy of rVHSV-A-IFNγ-ΔG that was improved by the additional expression of IFNγ.
To generate single-cycle live virus having cross-genotype protection ability, in this study, the rVHSV-Ia GΔTM single-cycle virus expressing the genotype Ia G gene, in which the transmembrane and C-terminal cytoplasmic regions are truncated based on rVHSV of genotype IVa, was rescued using IVa G and Ia G protein-expressing EPC cell line. Rescued viruses showed a high protective effect through the immunization test against wild-type VHSV genotype Ia and induced high antibody titer against VHSV genotype IVa and Ia.
To compare the cross-protective ability of vaccine types, immunization tests and antibody analysis were conducted using DNA vaccine, FKC vaccine, and rVHSV-ΔG single-cycle live virus. Although rVHSV-ΔG single-cycle live virus was not the form of vaccine that induces the most neutralizing antibodies, it showed very high cross-protective ability against VHSV genotype Ia and IHNV. The cross-protection mechanism of single-cycle live virus was analyzed through RNA-seq of immunization with DNA vaccine, FKC vaccine and rVHSV-ΔG single-cycle live virus rescued based on VHSV genotype IVa in rainbow trout. RNA-seq results from the rVHSV-ΔG single-cycle live virus-based vaccinated group, higher gene expression changes were confirmed on day 1, 3, and 28 compared to DNA and FKC vaccinated groups. In DEG analysis, various immune genes were expressed in rVHSV-ΔG single-cycle live virus vaccinated group. In particular, it was confirmed that T cell-related immune genes were consistently up-regulated from early phase to late phase of vaccination. These results are considered to be used as a useful vaccine for T cell-mediated cross-protection against heterologous viruses as well as in the form of a safe vaccine as a live-attenuated virus vaccine for VHSV because single-cycle live virus does not have the replication ability.
- Issued Date
- 2022
- Awarded Date
- 2022. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 수산생명의학과
- Advisor
- 김기홍
- Table Of Contents
- Part I: Replication-competent VHSVs 1
Chapter Ⅰ. Compensatory mutations in recombinant VHSVs engineered to mutate two amino acids (D62A-E181A) in matrix protein of VHSV genotype IVa 1
1. Introduction 2
2. Materials and methods 4
2.1 Cells and virus 4
2.2 Generation of M gene-mutated recombinant viruses 6
2.3 Comparison of cytopathic effect and virus growth using M gene-mutated viruses 6
2.4 Quantitation of Mx1 and NFkB gene expression in EPC cells 6
2.5 DNA fragmentation of EPC cells by M gene-mutated rVHSVs infection 7
2.6 Statistical analysis 7
3. Results 10
3.1 Generation of M gene-mutated rVHSVs 10
3.2 Replication ability of M gene-mutated rVHSVs 13
3.3 Cytopathic effect in EPC cell infected M gene-mutated rVHSVs 14
3.4 Quantitation of Mx1 and NFkB gene expression in EPC cells using luciferase reporter system 16
3.5 Effect of DNA fragmentation by M gene-mutated recombinant viruses infection 19
4. Discussion 21
5. Reference 25
Chapter Ⅱ. Generation of Type I interferon expressing recombinant VHSVs and application for attenuated vaccine 30
1. Introduction 31
2. Materials and methods 33
2.1 Cells and virus 33
2.2 Cloning of olive flounder type I interferons cDNAs 33
2.3. Generation of OFIFN4-expressing recombinant VHSVs 34
2.4. Verification of OFIFN4-expressing recombinant VHSVs 35
2.5. Comparison of growth using OFIFN4-expressing recombinant VHSVs 35
2.6 Comparison of cytopathic effect in OFIFN4-expressing EPC cell line 36
2.7 Analysis of the Mx1 gene expression in EPC and HINAE cells 36
2.8 Immunization with OFIFN4-expressing recombinant VHSVs 37
2.9 Statistical analysis 37
3. Results 39
3.1. Detection and cloning of OFIFN4 gene ORF 39
3.2. Generation and growth analysis of rVHSV-A-OFIFN4 and rVHSV-A-OFIFN4ΔNV 40
3.3. Effect of OFIFN4 expression in EPC cells on resistance against VHSV 43
3.4 Analysis of interferon realted Mx1 gene expression in EPC and HINAE cells 44
3.5 Vaccine potential of rVHSV-A-OFIFN4 and rVHSV-A-OFIFN4 ΔNV 45
4. Discussion 46
5. Reference 48
Chapter Ⅲ. Generation of Olive flounder C-type lectin-expressing VHSV and its effect on respiratory burst activity of phagocytes 52
1. Introduction 53
2. Materials and methods 55
2.1. Cloning for OFCTL and OFCTLepn 55
2.2. Cell and virus 56
2.3. Generation of rVHSV-A-OFCTL and rVHSV-A-OFCTLepn 56
2.4. Confirmation of rVHSV-A-OFCTL and rVHSV-A-OFCTLepn 57
2.4. Sample preparation and preparation of peripheral-blood phagocyte 57
2.5 Chemiluminesence (CL) assay 58
2.6 Statistical analysis 58
3. Results 60
3.1. Cloning of OFCTL and OFCTLepn 60
3.2. Generation of rVHSV-A-OFCTL and rVHSV-A-OFCTLepn 63
3.3. Respiratory burst activity by OFCTL expressing rVHSV 65
4. Discussion 66
5. Reference 68
Part II: Replication-incompetent VHSVs 72
Chapter Ⅳ. Protection of olive flounder (Paralichthys olivaceus) against VHSV by immunization with single-cycle recombinant VHSV expressing interferon-γ 72
1. Introduction 73
2. Materials and methods 75
2.1 Cells and virus 75
2.2 Rescue of single-cycle rVHSV-A-IFNγ-ΔG 75
2.3 Verification of rVHSV-A-IFNγ-ΔG using RT-PCR 76
2.4 Confirmation of rVHSV-A-IFNγ-ΔG single-cycle ability to produce infective viral particles using plaque assay 76
2.5 Immunization and challenge 77
2.6 ELISA 78
2.7 Statistical analysis 80
3. Results 80
3.1 Generation of single-cycle rVHSV-A-IFNγ-ΔG 80
3.2 Analysis of rVHSV-A-IFNγ-ΔG ability to produce infective viral particles 82
3.3 Immunization and challenge 83
3.4 Detection of VHSV-specific antiviral activity by ELISA 84
4. Discussion 85
5. Reference 88
Chapter Ⅴ. Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia by immunization with VHSV genotype IVa-based single-cycle viruses 90
1. Introduction 91
2. Materials and methods 94
2.1 Cells and virus 94
2.2 Establishment of Ia G protein-expressing EPC cells 94
2.3 Generation of rVHSV-Ia GΔTM recombinant viruses 95
2.4 Verification of rVHSV-Ia GΔTM and ability to produced infective viral particles 96
2.5 Immunization and challenge 97
2.6 Neutralization test by ELISA 97
2.7 Statistical analysis 98
3. Results 100
3.1 Establishment of Ia G protein-expressing EPC cells 100
3.2 Generation of rVHSV-Ia GΔTM single-cycle viruses 101
3.3 Analysis of rVHSV-Ia GΔTM ability to produce infective viral particles 102
3.4 Vaccine potential of rVHSV-Ia GΔTM 103
3.5 ELISA 105
4. Discussion 107
5. Reference 111
Chapter Ⅵ. Differences in cross protective ability among VHSV genotype IVa-based single-cycle live vaccine, inactivated vaccine and DNA vaccine against VHSV genotype Ia and IHNV 115
1. Introduction 116
2. Materials and methods 118
2.1 Cells and virus 118
2.2 Preparation of Vaccines and immunization test 118
2.3 Antibody titer analysis by ELISA 119
2.4 Immunization for RNA-seq 120
2.5 RNA preparation and construction of cDNA libraries for RNA-seq 120
2.6 RNA-sequencing and data analysis differential gene expression 121
2.7 Functional Annotation and Enrichment analysis 121
2.8 Statistical analysis 122
3. Results 123
3.1 The cross-protective effect of DNA vaccine and the analysis of antibody titer by ELISA 123
3.2 The cross-protective effect of FKC vaccine and the analysis of antibody titer by ELISA 126
3.3 The cross-protective effect of rVHSV-ΔG single-cycle live virus and the analysis of antibody titer by ELISA 129
3.4 Statistical summary of RNA-seq 132
3.5 Identification of RNA-seq after immunization of vaccines on 1day 134
3.5.1 DEGs analysis on 1day 134
3.5.2 Gene ontology (GO) analysis on 1day 136
3.5.3 Screening and comparison of immune-related DEGs on 1day 140
3.6 Identification of RNA-seq after immunization of vaccines on 3day 145
3.6.1 DEGs analysis on 3day 145
3.6.2 Gene ontology (GO) analysis on 3day 147
3.6.3 Screening and comparison of immune-related DEGs on 3day 151
3.7 Identification of RNA-seq after immunization of vaccines on 28day 155
3.7.1 DEGs analysis on 28day 155
3.7.2 Gene ontology (GO) analysis on 28day 157
3.7.3 Screening and comparison of immune-related DEGs on 28day 161
4. Discussion 165
5. Reference 171
Abstract 176
Acknowledgment 179
- Degree
- Doctor
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