Title Page
Contents
ABSTRACT 9
1. Introduction 11
1.1. Japanese encephalitis virus 11
1.2. The structure and life cycle of JEV 14
1.3. Japanese encephalitis vaccine 18
1.4. Reverse genetics system 21
2. Materials & Methods 24
2.1. Cell lines 24
2.2. Viruses 24
2.3. Construction of chimeric virus 24
2.4. Quantitative RT-PCR (qRT-PCR) 25
2.5. Plaque assay 25
2.6. Western blot assay 26
2.7. Reverse Transcription polymerase chain reaction (RT-PCR) 26
2.8. Sequence analysis 27
2.9. Animals 27
2.10. Immunization of JEV in mice 27
2.11. Enzyme-Linked Immunosorbent Assay (ELISA) 27
2.12. Micro-neutralization assay 28
2.13. Statistical analysis 28
3. Results 34
3.1. Construction of an infectious, full-length cDNA clone of chimeric JEV 34
3.2. Construction and confirmation of chimeric JEV 38
3.3. Growth characteristics of chimeric JEV 43
3.4. Pathogenicity of the chimeric JEV Strain in Mice 45
3.5. Immunogenicity of the rJEV-G1 Strain in BALB/C mice 47
3.6. Protective capacity the of G1 K05GS strain in rJEV-G1 vaccinated mice 49
4. Discussion 52
References 54
Abstract (in Korean) 58
Table 1. Japanese encephalitis vaccine type 20
Table 2. Primers used for amplification of the envelope (E) gene and backbone of Japanese encephalitis virus 32
Table 3. Primers used for RT-PCR 33
Table 4. Mutations in rJEV-G1 clone as compared to parental virus 42
Table 5. Survival rates in BALB/c Mice 51
Figure 1-1. The emergence time of Japanese encephalitis virus genotype and number of clinical cases in Korea, 2012-2022 13
Figure 1-2. Genome structure of the Japanese encephalitis virus 15
Figure 1-3. Japanese encephalitis virus transmission pathways 16
Figure 1-4. Japanese encephalitis virus replication 17
Figure 1-5. Reverse genetic system establishment of the reverse genetics for Japanese encephalitis virus 23
Figure 2-1. Molecular cloning strategy overview 29
Figure 2-2. Nucleotide sequences of codon-optimized JEV prM/E protein 30
Figure 2-3. Experimental design of mouse immunization and challenge 31
Figure 3-1. Schematic representation of the chimeric construct JEV 35
Figure 3-2. Agarose gel electrophoresis of products 36
Figure 3-3. Sequence analysis of protein coded by gene inserted in the rJEV-G1 plasmid 37
Figure 3-4. JEV-specific cytopathic effect (CPE) of rescued viruses 39
Figure 3-5. Characterization of the chimeric JEV expressing E genes 40
Figure 3-6. RT-PCR analysis of chimeric JEV genome 41
Figure 3-7. Growth properties of chimeric and parental JEV strains 44
Figure 3-8. Murine neurovirulence of chimeric JEV strains 46
Figure 3-9. Immunogenicity of the rJEV-G1 strain in vaccinated BALB/c mice 48
Figure 3-10. G1 K05GS strain challenge test in immunized mice 50