日本脑炎病毒基因Ⅰ型弱毒株鉴定
发布时间：2024-02-29 22:06
日本脑炎(Japanese encephalitis,JE)是由日本脑炎病毒(Japanese encephalitis virus JEV)感染引起的人畜共患传染病。通过疫苗免疫接种,能够预防JE。前期研究发现,JEV优势基因型由基因III型转变为基因I型,而基因III型来源的疫苗不能够完全保护基因I型JEV感染,需要研发基因I型疫苗。为此,开展了如下研究。为了鉴定细胞传代致弱的基因I型弱毒株SD12-F120,在体外和体内比较了SD12-F120株与亲本强毒株SD12的表型和基因型差异。SD12-F120在BHK-21细胞上形成了较小的病毒噬斑,且在小鼠原代神经元细胞和小鼠脑中的复制能力降低。通过腹腔或脑内途径接种高达105 PFU的SD12-F120,小鼠均未表现出JEV感染的临床症状,表明SD12-F120的神经侵袭力和神经毒力明显降低。SD12-F120免疫小鼠后,使用SD12攻毒,免疫小鼠未出现临床症状,获得全面保护。比较分析了SD12-F120和SD12间核苷酸序列差异,共发现29个核苷酸突变,其中20个属于无义突变,9个突变导致8个氨基酸变异,其中...
【文章页数】：102 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
英文缩略表
Chapter1 Introduction
1.1 Epidemiology and clinical presentation of Japanese Encephalitis virus infection
1.2 Japanese encephalitis virus genome organization and pathogensis
1.3 Genotype shift phenomenon
1.4 Disease Transmission
1.5 Diagnosis
1.6 Treatment
1.7 Prevention by Vaccination
1.7.1 Mouse brain derived inactivated Japanese encephalitis vaccine
1.7.2 Cell culture derived live-attenuated vaccine using SA14-14-2 strain
1.7.3 Cell culture derived-killed inactivated vaccine
1.7.4 Chimeric YFV-17D/JEV vaccine
1.8 Mice is a well-established animal model for JEV virulence
1.9 Study Objectives
CHAPTER2 PHENOTYPIC AND GENOTYPIC COMPARISON OF A LIVE-ATTENUATED JAPANESE ENCEPHALITIS VIRUS SD12-F120 STRAIN WITH ITS VIRULENT PARENTAL SD12 STRAIN IN VITRO AND VIVO
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Technical Route
2.3.2 Ethics statement
2.3.3 Viruses and cells
2.3.4 Passage of SD12 on BHK-21 cells
2.3.5 Plaque size determination
2.3.6 Neuroinvasiveness and neurovirulence tests in mice
2.3.7 Vaccination and challenge
2.3.8 Detection of JEV replication in mouse brain
2.3.9 Detection of expression of NS1 and NS1?in BHK-21 cells
2.3.10 Viral genome sequencing
2.3.11 Sequence analysis
2.3.12 Statistical analysis
2.4 Results
2.4.1 Differences in neuroinvasiveness and neurovirulence between the attenuated SD12-F120 and its virulent parental SD12 strains in mice
2.4.2 Protective efficacy of the attenuated SD12-F120 strain against its virulent parental SD12 strain challenge in mice
2.4.3 Reduced replication efficiency of the attenuated SD12-F120 strain in mouse brain and mouse primary neuron cells
2.4.4 Replication phenotype in BHK-21 cells
2.4.5 Overview of nucleotide and amino acid variations between the attenuated SD12-F120 and its virulent parental SD12 strains
2.4.6 Comparison of amino acid substitutions in E protein among five isogenic attenuated and virulent strain pairs
2.4.7 Comparison of variations in other regions among five isogenic attenuated and virulent strain pairs
2.5 Discussion
2.6 Conclusion
CHAPTER3 ADAPTATION OF A LIVE-ATTENUATED GENOTYPE I JAPANESE ENCEPHALITIS VIRUS TO VERO CELLS IS ASSOCIATED TO MUTATIONS IN STRUCTURAL GENES
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Technical Route
3.3.2 Ethics statement
3.3.3 Viruses and cells
3.3.4 Passage of SD12 on Vero cells
3.3.5 Plaque titration and purification
3.3.6 Neuroinvasiveness and Neurovirulence Tests in Mice
3.3.7 Vaccination and Challenge
3.3.8 Detection of neutralizing antibody titers
3.3.9 Complete genome sequencing
3.3.10 Statistical analyses
3.4 Results
3.4.1 Growth properties of Vero cell adapted SD12-F120VC Vaccine variants
3.4.2 Attenuation Phenotype
3.4.3 Differences in Neuroinvasiveness and Neurovirulence of SD12-F120VC variants
3.4.4 Protective efficacy of the SD12-F120VC strain against GI and GIII challenge in Mice
3.4.5 Protective efficacy of the SA14-14-2 strain against GI and GIII challenge in Mice
3.4.6 Neutralization antibody
3.4.7 Genomic analysis
3.5 Disscussion
3.6 Conclusion
References
Acknowledgement
附件
本文编号：3914994
【文章页数】：102 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
英文缩略表
Chapter1 Introduction
1.1 Epidemiology and clinical presentation of Japanese Encephalitis virus infection
1.2 Japanese encephalitis virus genome organization and pathogensis
1.3 Genotype shift phenomenon
1.4 Disease Transmission
1.5 Diagnosis
1.6 Treatment
1.7 Prevention by Vaccination
1.7.1 Mouse brain derived inactivated Japanese encephalitis vaccine
1.7.2 Cell culture derived live-attenuated vaccine using SA14-14-2 strain
1.7.3 Cell culture derived-killed inactivated vaccine
1.7.4 Chimeric YFV-17D/JEV vaccine
1.8 Mice is a well-established animal model for JEV virulence
1.9 Study Objectives
CHAPTER2 PHENOTYPIC AND GENOTYPIC COMPARISON OF A LIVE-ATTENUATED JAPANESE ENCEPHALITIS VIRUS SD12-F120 STRAIN WITH ITS VIRULENT PARENTAL SD12 STRAIN IN VITRO AND VIVO
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Technical Route
2.3.2 Ethics statement
2.3.3 Viruses and cells
2.3.4 Passage of SD12 on BHK-21 cells
2.3.5 Plaque size determination
2.3.6 Neuroinvasiveness and neurovirulence tests in mice
2.3.7 Vaccination and challenge
2.3.8 Detection of JEV replication in mouse brain
2.3.9 Detection of expression of NS1 and NS1?in BHK-21 cells
2.3.10 Viral genome sequencing
2.3.11 Sequence analysis
2.3.12 Statistical analysis
2.4 Results
2.4.1 Differences in neuroinvasiveness and neurovirulence between the attenuated SD12-F120 and its virulent parental SD12 strains in mice
2.4.2 Protective efficacy of the attenuated SD12-F120 strain against its virulent parental SD12 strain challenge in mice
2.4.3 Reduced replication efficiency of the attenuated SD12-F120 strain in mouse brain and mouse primary neuron cells
2.4.4 Replication phenotype in BHK-21 cells
2.4.5 Overview of nucleotide and amino acid variations between the attenuated SD12-F120 and its virulent parental SD12 strains
2.4.6 Comparison of amino acid substitutions in E protein among five isogenic attenuated and virulent strain pairs
2.4.7 Comparison of variations in other regions among five isogenic attenuated and virulent strain pairs
2.5 Discussion
2.6 Conclusion
CHAPTER3 ADAPTATION OF A LIVE-ATTENUATED GENOTYPE I JAPANESE ENCEPHALITIS VIRUS TO VERO CELLS IS ASSOCIATED TO MUTATIONS IN STRUCTURAL GENES
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Technical Route
3.3.2 Ethics statement
3.3.3 Viruses and cells
3.3.4 Passage of SD12 on Vero cells
3.3.5 Plaque titration and purification
3.3.6 Neuroinvasiveness and Neurovirulence Tests in Mice
3.3.7 Vaccination and Challenge
3.3.8 Detection of neutralizing antibody titers
3.3.9 Complete genome sequencing
3.3.10 Statistical analyses
3.4 Results
3.4.1 Growth properties of Vero cell adapted SD12-F120VC Vaccine variants
3.4.2 Attenuation Phenotype
3.4.3 Differences in Neuroinvasiveness and Neurovirulence of SD12-F120VC variants
3.4.4 Protective efficacy of the SD12-F120VC strain against GI and GIII challenge in Mice
3.4.5 Protective efficacy of the SA14-14-2 strain against GI and GIII challenge in Mice
3.4.6 Neutralization antibody
3.4.7 Genomic analysis
3.5 Disscussion
3.6 Conclusion
References
Acknowledgement
附件
本文编号：3914994
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