日本腦炎(Japanese encephalitis,JE)是由日本腦炎病毒(Japanese encephalitis virus JEV)感染引起的人畜共患傳染病。通過(guò)疫苗免疫接種,能夠預(yù)防JE。前期研究發(fā)現(xiàn),JEV優(yōu)勢(shì)基因型由基因III型轉(zhuǎn)變?yōu)榛騃型,而基因III型來(lái)源的疫苗不能夠完全保護(hù)基因I型JEV感染,需要研發(fā)基因I型疫苗。為此,開(kāi)展了如下研究。為了鑒定細(xì)胞傳代致弱的基因I型弱毒株SD12-F120,在體外和體內(nèi)比較了SD12-F120株與親本強(qiáng)毒株SD12的表型和基因型差異。SD12-F120在BHK-21細(xì)胞上形成了較小的病毒噬斑,且在小鼠原代神經(jīng)元細(xì)胞和小鼠腦中的復(fù)制能力降低。通過(guò)腹腔或腦內(nèi)途徑接種高達(dá)10⁵ PFU的SD12-F120,小鼠均未表現(xiàn)出JEV感染的臨床癥狀,表明SD12-F120的神經(jīng)侵襲力和神經(jīng)毒力明顯降低。SD12-F120免疫小鼠后,使用SD12攻毒,免疫小鼠未出現(xiàn)臨床癥狀,獲得全面保護(hù)。比較分析了SD12-F120和SD12間核苷酸序列差異,共發(fā)現(xiàn)29個(gè)核苷酸突變,其中20個(gè)屬于無(wú)義突變,9個(gè)突變導(dǎo)致8個(gè)氨基酸變異,其中...

【文章頁(yè)數(shù)】：102 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
Abstract
英文縮略表
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



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