臺灣博碩士論文加值系統

1998年夏天，台灣地區爆發了腸病毒的流行，根據調查此次流行的工作小組所報告，在這波流行期間共有129,106個發生手足口症或咽峽炎的病例，其中有405個病人的病情較為嚴重，合併有腦炎、無菌性腦膜炎、肺水腫、肢體麻痺，或心肌炎等症狀，死亡人數累計達到78人，多為5歲以下患者。腸病毒為正性單股核醣核酸病毒，屬於小RNA病毒科（Picornaviridae），目前已知會感染人類的此類病毒共有66種，包括小兒麻痺病毒1-3型（Poliovirus type 1-3）、A型克沙奇病毒（Coxsackievirus A group）、B型克沙奇病毒（Coxsackievirus B group）、人類腸道致病性病毒（Echoviruses）及其他腸病毒型（Enterovirus type 68-71）。通常大部分腸病毒的感染症狀輕微，但是也有些會造成嚴重的疾病甚至死亡，導致1998年台灣地區嚴重腸病毒疫情的腸病毒71型，即為一例。
腸病毒的血清型眾多，發展通用疫苗誠屬不易，本研究主要致力於了解腸病毒蛋白酵素2A的特性，以做為將來發展抗腸病毒藥物之理論基礎。我們取得了1998年台灣腸病毒流行期間的十株腸病毒71型臨床分離株、1969年所分離的EV71/BrCr原型以及1993到1999年所分離的十株克沙奇病毒B1型臨床病毒株，進行腸病毒蛋白酵素2A之研究。針對蛋白酵素2A的區域，以RT-PCR的方法，分析其DNA核酸與蛋白質氨基酸序列，結果發現，腸病毒71型各株序列相差無幾而克沙奇病毒B1型各株則變異較大。因此，在腸病毒71型方面，我們繼續以原型株分析其蛋白酵素2A之切點，採用點突變的方法，在原核以及真核細胞表現系統皆已證實其切點位置；而在克沙奇病毒B1型方面，無論在非洲綠猴腎細胞或人類神經膠質母細胞瘤之多形性細胞中，皆觀察到臨床株CVB1-8之複製速度或細胞病變比CVB1-5來得快，並且其對宿主細胞蛋白eIF4G的切割亦較迅速，種種證據顯示，兩株臨床株在蛋白質2A序列上的差異，的確可能影響兩者在感染速度之快慢，值得再進一步深入探討。

An outbreak associated with enterovirus infection occurred in Taiwan area during the year of 1998. It was reported that there were 129,106 cases of hand-foot-and-mouth disease or herpangina in the outbreak ; among them, 405 patients suffered from severe diseases with complications including encephalitis, aseptic meningitis, pulmonary edema, hemorrhage, acute flaccid paralysis, and myocarditis. Accumulated death toll has been 78 patients ; most of them were five years old or younger.
Enteroviruses belong to the Family Picornaviridae, where the picornaviral genome consists of a single strand of message-active RNA. Presently, it is known that there are 66 types that can infect human, including Polioviruses type 1-3, Coxsackieviruses A group, Coxsackieviruses B group, Echoviruses and Enteroviruses type 68-71. Most of the syndromes caused by enterovirus infections are mild, with few exceptions such as enterovirus 71 (EV71) where severe diseases even death may incur.
It will be challenging to develop vaccines against enteroviruses due to the multiple serotypes. Therefore, an effort has been made to study the protease 2A that plays a major role in viral replication, and may serve as a molecular target for anti-enterovirus therapy. We started by analysis of the protease 2A in 10 EV71 isolates from the 1998 outbreak and 10 Coxsackie B1 (CVB1) strains collected during 1993-1999. Analysis sequences of the DNA nucleotide and protein amino acid showed that the variations among the EV71 are slight whereas those of CVB1 are greater. We further map the cleavage site of the protease 2A in EV71 prototypic strain (BrCr) by molecular cloning followed by site-directed mutagenesis. The cleavage site was confirmed by both prokaryotic and eukaryotic expression systems. As for the study on the CVB1, two clinical isolates, CVB1-8 and CVB1-5, with relatively greater variations in the protease 2A were used for further investigation. It was shown that the rate of viral replication and cytopathic effect is more rapid with the CVB1-8 infection than those with the CVB1-5 in the Vero and GBM cells. This is consistent with the finding that the cleavage of the host protein-eIF4G by protease 2A is more effective with CVB1-8 infection. The data indicate that the variations in the protease 2A regions between these two CVB isolates may account for the differences in their replication and pathogenesis. Experiments to clarify the mechanism are in progress.

摘要……………………………………………………….1
緒論……………………………………………………….6
基本材料與實驗方法……………………………………22
結果……………………………………………………….45
討論……………………………………………………….62
參考文獻………………………………………………….68
表圖……………………………………………………….74
附錄………………………………………………………116

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