日本腦炎病毒是屬於黃病毒科(Flaviviridae)中黃病毒屬(Flavivirus)的病毒，是一種經由蚊子傳播給人類的節肢動物攜帶病毒。日本腦炎病毒感染人類會引起嚴重且具高致死率的疾病，一半以上的存活者會導致永久性神經損壞。日本腦炎病毒臺灣弱毒株CH2195LA是由臺灣本土株日本腦炎病毒CH2195中分離出，具有會被中和性單株抗體E3.3作用的特性，研究中用來與臺灣弱毒株CH2195LA進行比較的非減毒株CH2195SA同樣是由CH2195中分離出，但是不會被中和性單株抗體E3.3作用。本研究目的在於分析臺灣弱毒株日本腦炎病毒CH2195LA的全長核酸序列基因型及其在不同細胞株的病毒繁殖複製型態。首先直接定序病毒cDNA片段，找出弱毒株CH2195LA與非減毒株CH2195SA之間基因組的差異，再利用核酸序列演化樹叢分析，比較弱毒株CH2195LA與SA14-14-2、SA14-2-8及RP-2ms等文獻報導的減毒株在內的其他13株日本腦炎病毒株的演化遺傳關係。基因型的研究結果顯示，弱毒株CH2195LA與非減毒株CH2195SA之間共有E-85、E-306、E-331、E-387、NS2A-215、NS3-350、NS4B-196、NS4B-197、NS4B-198等9個胺基酸相異點，而弱毒株CH2195LA與經過鼠腦繼代四代的CH2195LA-SM4之間共有結構蛋白部分M-73、M-80、E-161、E-170、E-276以及非結構蛋白部分NS2A-136、NS2A-215、NS3-346、NS4A-128、NS4B-196、NS4B-197、NS4B-198等12個胺基酸相異點。核酸序列演化樹叢圖顯示弱毒株CH2195LA、非減毒株CH2195SA與JaOArS982屬於同一病毒亞群，但是弱毒株CH2195LA與SA14-14-2、SA14-2-8及 RP-2ms三株減毒株在演化遺傳關係上並不相近。
臺灣弱毒株日本腦炎病毒CH2195LA細胞株複製型態的研究，先比較出弱毒株CH2195LA與非減毒株CH2195SA在8株不同細胞株的病毒複製型態差異，再以相對適性分析比較毒力不同的CH2195LA與CH2195SA等比例混合後感染Vero、THP-1、C6/36等三株細胞株的繁殖複製競爭力。細胞株複製型態的研究結果顯示，弱毒株CH2195 LA只有在Vero細胞株中的繁殖速度及最大感染力比非減毒株CH2195SA高。由相對適性向量分析發現，弱毒株CH2195 LA只有在Vero細胞持續繼代感染後出現適性增加的趨勢，而鼠腦繼代後的CH2195LA-SM4在Vero細胞持續繼代感染後出現適性增加但是較弱毒株CH2195LA低的趨勢。本篇論文比較出臺灣弱毒株日本腦炎病毒CH2195LA的毒力遺傳決定位可能是分佈在E、NS2A、NS3、NS4A、NS4B等多個基因中，並且得到弱毒株CH2195LA基因組經過鼠腦繼代後並不穩定，但是適合在Vero細胞株繁殖複製的結果，期能有助於提供研究日本腦炎病毒毒力及發展更好的日本腦炎減毒疫苗的資訊。

Japanese encephalitis virus (JEV) is a mosquito-borne virus that belongs to the family Flaviviridae, genus Flavivirus. JEV causes fatal disease in human infection case and leads half of the survivors to neural system disrupted and psychoneurosis. The attenuated variant CH2195LA and the non-attenuated variant CH2195SA of Japanese encephalitis virus were both isolated from wild-type Taiwanese mosquito isolates CH2195. The attenuated variant CH2195LA was sensitive to mAb E3.3 neutralization and the non-attenuated variant CH2195SA was resistant to mAb E3.3 neutralization. The purpose of this research is to analyze the complete nucleotide sequence genotype and cell-line multiplication pattern phenotype of the attenuated variant CH2195LA of Japanese encephalitis virus. In the genotype study , we determined the complete nucleotide sequences of the two variants and found amino acid changes between them. Then we used complete nucleotide sequences phylogenetic analysis to compare the evolutionary relationship of the attenuated variant CH2195LA and other 14 JEV strains. Our study found there were 9 amino acid changes between the complete sequences of the two variants, including E-85, E-306, E-331, E-387, NS2A-215, NS3-350, NS4B-196, NS4B-197, and NS4B-198. There were 12 amino acid changes between CH2195LA and CH2195LA-SM4, including M-73, M-80, E-161, E-170, E-276, NS2A-136, NS2A-215, NS3-346, NS4A-128, NS4B-196, NS4B-197, and NS4B-198. The phylogenetic analysis indicated the attenuated variant CH2195LA is closely related to the non-attenuated variant CH2195SA and JaOArS982, and is distantly related to SA14-14-2, SA14-2-8, and RP-2ms of the attenuated JEV strains.
The multiplication patterns of the two variants were investigated in eight different cell lines. The virus replication rate and maximum infectivity of the attenuated variant CH2195LA in Vero cell multiplication pattern were both higher than those of the non-attenuated variant CH2195SA. The multiplication patterns for CH2195LA compared with CH2195SA in other seven cell lines were different from Vero cell. We further determined the relative fitness of CH2195LA verse CH2195SA by using direct competitive mixture of both variants to measure the relative fitness change in three different cell lines passages, including Vero, C6/36, and THP-1. Relative fitness vector plot indicated the attenuated variant CH2195LA showed increased viral fitness in Vero cell line passages, but viral fitness of the mouse brain passage sample CH2195LA-SM4 was lower than the attenuated variant CH2195LA. The multiplication patterns and relative fitness results indicated that the attenuated variant CH2195LA was unique to culture in Vero cell lines, but it changed after mouse brain passage. The results suggested that the genome of the attenuated variant CH2195LA was not stable after mouse brain passaged. We can raise its gemome stability by continuous passaging the attenuated variant CH2195LA in Vero cell line. The results of this research were important for realizing the attenuation mechanism of the attenuated variant CH2195LA and developing attenuated JEV vaccines.

英文摘要……………………………………………………….3

第一章 緒論………………………………………………….5

第二章 材料與方法……………………………….………………22

第三章 結果……………………………………….………………36

第四章 討論……………………………………….………………46

第五章 結論………………………………………………..58

圖表…………………………………………………………60

參考文獻……………………………………………………….…..81

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