臺灣博碩士論文加值系統

腸病毒71型在分類上屬於小RNA病毒科，具有一條正股的RNA基因，可以被轉譯出一條含有2,193個胺基酸的聚合蛋白。一旦病毒感染宿主細胞後，這條前驅蛋白質就會被切割成4個結構蛋白(VP1、VP2、VP3 與VP4)及7個非結構蛋白(2A、2B、2C、3A、3B、3C與3D)。自從1969年，在美國加州腸病毒71型之基因型A第一次被分離出來後，世界各地陸續都有幾波疫情爆發。根據腸病毒71型流行病學的研究可知，在1997年之後，基因型B3-B5以及C2-C5都曾在亞太地區造成嚴重的流行。而在1998年，基因型C2之腸病毒71型造成台灣第一次的大規模流行，在405例重症病例中有78名小朋友死亡。此外，在2008年與2012年則是基因型B5的病毒株造成規模不小之手足口症的流行。因此，本研究之目的是分析1998年、2008年與2012年來自輕症或重症之臨床病毒株的全長序列，希望藉此找出與疾病嚴重程度有關聯之遺傳變異位點。我們首先進行2008年或2012年之輕重症的比較，在輕症與重症間都沒有發現具有顯著差異的位點；然而將2008年與2012年全長序列作比較，則發現2012病毒株在非結構蛋白區有許多胺基酸改變之位點，這可能與B5病毒株繼2008年之後，又再次於2012年爆發有關。1998年，我們總共收集了9例死亡病例與15例非死亡病例，產生感染性克隆後再送定序，結果發現在2C的第302個胺基酸與3C的第55個胺基酸在死亡病例與非死亡病例中有所差異。在死亡病例病毒分離株的序列中，2C 302的位點傾向為白胺酸(L)多於纈胺酸(V)，且3C 55的位點傾向是纈胺酸(V)多於異白胺酸(I)；在非致死病例中這兩點的胺基酸出現頻率則和致死病例相反。而死亡病例中，白胺酸(L)和纈胺酸(V)同時出現的比率有56%；在非死亡病例中只有27%。為了瞭解2C 302與3C 55之位點在功能上所扮演的角色，我們利用感染性克隆之定點突變的方式，產生具有2C 302或3C 55之單點與雙點突變的重組病毒(2CL302V、3CV55I、2CL302V3CV55I)，並觀察病毒突變前後的生物特性。結果顯示突變前後病毒的溫度感受性與使細胞凋亡的能力沒有太大差異；但在生長速度方面，2CL3023CV55病毒株在高MOI的情況下，在RD 與SK-N-SH兩種細胞中複製的速度都比具有2CV302、3CI55、2CV3023CI55病毒株的病毒快，而且2CL3023CV55也會產生較大型的病毒溶斑。此外，先前有許多研究已經證實，一氧化氮可抑制多種病毒與細菌的生長，包括腸病毒71型；我們的實驗也看到腸病毒71型被一氧化氮抑制的情況，且2C 302及3C 55之位點突變會改變對一氧化氮之感受性。綜合以上論述，我們認為2C 302與3C 55之位點在腸病毒71型複製的不同階段扮演不同的角色。

Enterovirus 71 (EV71) is a non-enveloped, positive-strand RNA virus of the Picornaviridae family. The viral genome encodes a single polyprotein precursor of 2,193 amino acids. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3 and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C and 3D) proteins. Several outbreaks were reported worldwide since it had been isolated firstly in California in 1969 known as EV71 genotype A. Studies on EV71 epidemiology showed that genotype B3-B5 and C2-C5 have caused large outbreaks in the Asia-Pacific region since 1997. During 1998, EV71 genotype C2 burst out the biggest outbreak in Taiwan, resulting in 405 cases of severe neurological sequelae and 78 deaths. In addition, in 2008 and 2012, genotype B5 has caused serious HFMD epidemics in Taiwan. In this study, we aim to find genetic variations between EV71 isolates in 1998, 2008 and 2012 by analyzing EV71 full-length sequences. First of all, we found no conserve difference among severe and mild cases in 2008 or 2012. Interestingly, there were several different amino acids in non-structure proteins between 2008 and 2012. These differences in non-structure proteins may correlate with the re-emergence of EV71 genotype B5 in 2012. In 1998, the 9 fatal and 15 non-fatal cases were used to produce full length clone and sequence. Two major nucleotide differences among fatal and non-fatal cases were identified at the position 4985 in 2C302 and position 5555 in 3C55. In fatal cases, leucine was identified more than valine at 2C302 and more valine than isoleucine was found at 3C55. In non-fatal cases, the frequency of amino acid expressing is opposite. The percentage of leucine and valine which expressed simultaneously in 2C302 and 3C55 is 56% among fatal cases and only 27% among non-fatal cases. In order to examine the functional role of 2C302 and 3C55, we introduced the 2C-L302V and 3C-V55I mutations, single or double, by site-directed mutagenesis to the EV71 full length infectious cDNA clone. Reverse genetics viruses (rg viruses) obtained were used to examine viral properties. The four rg viruses all showed temperature sensitive phenotype and induced similar effect of apoptosis. However, the rg virus 6359, which contained 2C-L302 / 3C-V55 showed faster growth than other mutant viruses on one step viral growth curve at high moi in both RD and SK-N-SH cells. In addition, the 6359 rg virus formed larger plaques compared to that of mutant rg viruses. In addition, it has been demonstrated that nitric oxide (NO) could inhibit the replication of many pathogens, including EV71. We found the 2C302 and 3C55 can affect the sensitivity of inhibition by NO. This study suggests amino acids at 2C302 and 3C55 play various roles in EV71 replication.

目錄
第一章 緒論 1
一、 腸病毒的分型 1
二、 腸病毒71型的構造與複製 3
三、 腸病毒71型的臨床病徵與流行病學 8
四、 腸病毒71型引起之免疫反應 13
五、 腸病毒71型的毒性決定基因研究 14
六、 腸病毒71型的生活史 17
七、 腸病毒71型之2C蛋白 20
八、 腸病毒71型之3C蛋白 22
九、 病毒之感染性克隆(infectious clone)系統 24
十、 研究動機及目標 25
第二章 材料與方法 28
一、 細胞與病毒 28
二、 感染性克隆(infectious clone)之建構 34
三、 定點突變(site direct mutagenesis) 43
四、 一氧化氮對病毒之影響 46
五、 重組分析(recombination analysis) 49
六、 親緣演化分析 (phylogenetic analysis) 50
七、 蛋白質結構預測軟體SWISS-MODEL 50
八、 統計分析 (Statistical analysis) 50
第三章 結果 51
一、 腸病毒71型臨床分離株之全長序列比較 51
1. 2008年與2012年之腸病毒71型病毒株全長序列的分析 52
2. 1998年大流行之腸病毒71型病毒株全長序列的分析 53
3. 1998年(基因型C2)、2008年與2012年(基因型B5)之腸病毒71型病毒株全長序列的比較 54
二、 腸病毒71型突變株之生物特性 55
1. 生長速率分析 56
2. 病毒溶斑型態分析 57
3. 溫度感受性分析 (temperature sensitivity) 57
4. 細胞凋亡分析 (apoptosis) 58
三、 一氧化氮對突變前後之腸病毒71型的抑制效果 59
1. 亞硝基乙醯青黴胺(SNAP)對細胞的毒性測試 59
2. SNAP在細胞內釋放NO的情形 60
3. 不同時間加入 SNAP 對腸病毒71型生長之影響 60
4. SNAP 對腸病毒71型生長曲線的影響 60
5. SNAP 對腸病毒71型突變株複製之影響 61
第四章 討論 62
參考文獻 70

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