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研究生:褚佩瑜
研究生(外文):Pei-Yu Chu
論文名稱:1998年臺灣地區腸病毒流行之研究
論文名稱(外文):Study of Enterovirus Infection in Taiwan in 1998
指導教授:彭健芳林貴香林貴香引用關係
指導教授(外文):Chien-Fang PengKuei-Hsiang Lin
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:159
中文關鍵詞:腸病毒手足口病種系分析分子流行病學
外文關鍵詞:enterovirusHand-foot and mouth diseasePhylogenetic analysisMolecular epidemiology
相關次數:
  • 被引用被引用:2
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  • 下載下載:72
  • 收藏至我的研究室書目清單書目收藏:3
在 1998年,臺灣全島暴發腸病毒的大流行,疫情震驚整個社會。數千名兒童感染了手足口病,導致七十八個死亡病例。因為人類腸病毒中各種不同的血清型會周而復始的流行著,而且它具有造成重大疾病的潛力,因此改進病毒診斷、鑑定流程及加強監督病毒感染是目前防治上的重大課題。為了著手於這個目標的探討,首先我們著重於病毒的監測、實驗室分離及診斷,以了解這波腸病毒流行的特質。
為了要了解1998年在臺灣爆發的腸病毒大流行在高雄地區的致病因,腸病毒的分離共選用 Vero, Hep-2, 及HeLa 細胞株,在培養細胞出現具腸病毒特徵的細胞病變效應,即進一步驟執行血清學及分子生物學的鑑定,包括間接螢光抗體試驗(indirect fluorescent antibody test,IFA)及微量中和試驗 (microneutralization test,NT)及RT-PCR。鑑定的出來的腸病毒,分別為 Enterovirus 71 (EV71,34.1%)、 Coxsackie A16(CA16,23.6%)、Echo 7(E7,17.1%)、polio Sabin strains(PS,11.4%)、Coxsackie B virus (CB,5.3%) 及其他未區別之腸病毒(EVs,8.5%)。在1998流行期間腸病毒的分離率呈現三個波分別在三月、六月及十月。三月病毒的分離率為21.1%(52/246)為高雄地區所獨有,主要致病病毒為E7及PS,疾病以呼吸道感染及中樞神經系統侵犯為主。手足口病的流行出現在五月到十二月間,有一主波(在六月,分離率30.4%, 75/246)、一副波(在九月)之特徵。致病病毒則以 EV71及 CA16為主。各不同來源檢體的腸病毒中分離率最高的是咽喉拭子(72.0 %)其次是糞便或直腸拭子的 12.2%及腦脊髓液的11%。各病毒分離率最高的檢體都是咽喉拭子,E7及PS自腦脊髓液的分離率(都是28.6%)顯著的高於EV71(4.8%,4/84)。最常被感染的年齡是一歲以下(32.4%),有 82.2% 的患者是五歲以下。 EV71與CA16很少感染六個月以下的嬰兒,而E7及PS感染的年齡成較廣,包括感染六個月以下的嬰兒。臨床病症上最常見的是手足口病 (HFMD,37.8%)、其他依次是呼吸道感染(28.0 %)、腦膜炎 (15.9%)、疹性咽峽炎 (3.7%),也發現到一些心肌炎(1.6%)的病例。EV71與CA16是手足口病的主要病因,其中只有EV71傾向造成中樞神經侵犯、可能導致重症及死亡病例。而E7及PS造成呼吸道感染及其中樞神經侵犯以腦膜炎為多。
分子序列及種系發生的分析已被廣泛的用來當濾過性病毒感染的分子診斷及分子流行病學工具。為了要在之下波感染腸病毒的基因特質,我們分析了腸病毒VP4基因上共207bp長度的序列,共分析了23株EV71及4 株 CA16。將結果與基因庫上已登錄的病毒序列做種系發生的比較。我們得到三個重要結論。第一、根據VP4基因序列分析我們有一大一小二個基因群的EV71 參與1998年腸病毒的大流行。 (1)小群的EV71 病毒株可能早在 1986 年就出現在臺灣的流行中,它們可能持續存在,也造成一些感染,然後在此次大流行再次出現。(2)在這次大流行中主要被分離出來的一群,應該是一新的變異株。第二、其VP4的核酸序列類似日本在1989年後分離的病毒株。而且死亡病例分離出來的病毒株分佈在這兩個基因群內,並沒有特殊臨床症狀上的區別。最後、我們的結果與其他根據 5''-NCR、VP1 或 VP4 基因之核酸序列做種系發生分析來研究 EV71 病毒株的結果類似,那就是沒發現有特殊的臨床症狀與特定的的基因型病毒株相關。但是我們的結果顯示以VP4區域基因序列分析的種系發生其結果與時間、地理緊緊相關。這區域的核酸序列對評估流行病毒的來源及流行病學的學研究上將會是個很好的工具。
The public epidemiological record in Taiwan astonished us by the outbreak of Enteroviruses infection in 1998. Thousands of children were suffering with Hand-Foot and Mouth Disease (HFMD). Seventy-eight children died of this outbreak of infection. When most of this infectious disease is under control, the evolution of this virus particle shifts the virulence from the non-pathogenic or mild pathogenic microbes to pathogenic ones. In addition, the ease of the traffic increases the spread of the infectious diseases. These newly emerging infectious agents have become the urgent issues of society today. Since the recurrence and potential to cause severe diseases for various serotypes of Enteroviruses, it mandates the need for improved surveillance and improved methods to identify the Enteroviruses in human disease. A series of studies were therefore undertaken to confirm the trait of causative agent for this infectious outbreak. We started our study with the clinical diagnosis and laboratory finding of the enteroviruses epidemic infection.
In order to understand the etiologic of the outbreak of Enteroviruses infections in Kaohsiung area, specimens were collected in 1998. Cell lines used for viral isolation were Vero, Hep-2, and HeLa. After CPE recognition, indirect fluorescent antibody tests (IFA) and neutralization test (NT) were used to identify the EVs. VP4 nucleotide sequences were analyzed by direct sequencing. Agreements of the results were found among these three identification methods. Eighty-four of the 246 EV isolates were EV71 (34.1%), Coxsackie A16 (23.6%), ECHO 7 (17.1%), polio Sabin strains (11.4%), Coxsackie B virus (5.3%), and others EVs (8.5%). About 30.4% (75/246) of the EV strains was isolated in June. Most of the viruses were isolated from throat swab (78.1 %) and 3.3% of them were isolated from cerebrospinal fluid. The most common age group was 1-month to 1-year old (33.8%). The most common feature was HFMD (37.8%). Respiratory tract infection (28.0 %), meningitis (15.9%), herpangina (3.7%), and myocarditis (1.6%) were also found in this study. Our results showed that EV71 and Coxsackie A 16 (CA16) were the major causative agents of HFMD, especially EV71 was responsible for the severe and fatal cases.
Nucleotide sequencing and phylogenetic analysis has been used extensively as epidemiological and diagnostic tools for the viruses’ infection. In order to understand the genetic characteristics of this infectious outbreak, we determined the 207-bp length of VP4 gene in 23 culture-proven EV71 isolates. We performed phylogenetic analysis based on these isolates of EV71 in Taiwan and upon 21 others isolated in GenBank. In summary, there are three important conclusions that can be obtained from our results. First based on the nucleotide sequences analysis, there were two genogroups responsible for part of the outbreak: (1) A relatively small cluster of viral strains may have been present in the 1986 Taiwan outbreak, which evolved. They were also responsible for part of the outbreak in 1998. (2) The major cluster of infectious strains that emerged in the most recent outbreak in Taiwan might be a new variant. Secondly, although our isolates yielded clinical similarity to the 1997 Malaysia outbreak, our VP4 nucleotide sequences were clustered into different genogroups (B vs. C-3). Finally, our results were similar to the findings of other studies in that they were unable to discern any relationship between clinical presentation and the genogroups origin of any EV71 strain, no matter what was based on the DNA sequencing for the region of 5-NCR, VP1 or VP4. It was noted that the severity and the clinical symptoms of EV71 in this outbreak were not linked to certain genogroups of VP4. However, the genogrouping of the EV71 region was closely correlated with various geographical distributions, which may be used as a useful tool for establishing epidemiological links. It was suggested that the phylogenetic analysis might provide one of the most efficient aids currently available for rapid tracking and further clarification of EV-infected specimens for epidemiological surveillance.
封面
目錄
中文摘要
英文摘要
誌謝
目錄
表目錄
圖目錄
英文專有名詞縮寫說明表
第一章 有關腸病毒流行之文獻回顧
一、 前言
二、 腸病毒的歷史背景與流行概況
三、 腸病毒的基本特性
四、 腸病毒常引引起的疾病與臨床症狀
五、 腸病毒感染之預後及影響其預後的因子
六、 腸病毒的致病機轉
七、 腸病毒感染的診斷
八、 治療與預防
第二章 1998 年高雄地區腸病毒流行致病原之研究
一、摘要
二、前言
三、材料與方法
四、結果
五、討論
第三章 VP4分子序列在腸病毒分型之研究
一、摘要
二、前言
三、材料與方法
四、結果
五、討論
第四章 1998 年臺灣流行腸病毒感染 71 之分子特性之研究
一、摘要
二、前言
三、材料與方法
四、結果
五、討論
第五章 展望與討論
一、前言
二、重系分析在腸病毒感染之研究
三、重建種系發生樹的方法
四、討論
參考資料
附錄
1. 小 RNA 病毒的分類
2. 腸病毒的基因組成
3. 小 RNA 病毒科核酸 5''-NCR 不完全互補的摺疊模型
4. 腸病毒的致病過程
5. 腸病毒的群感染造成全身器官症狀一覽表
6. 1980-1997 年間臺大醫學院、臺北榮民總醫院及高雄醫學大學附設醫院腸病毒之分離報告(表)
7. 1980-1997 年間臺大醫學院、臺北榮民總醫院及高雄醫學大學腸病毒之分離報告(圖)
8. 1990-1999 年 高雄醫學大學腸病毒之分離報告
9. 腸病毒 5''-NCR 高度保留區的SSCP結果
10. 演化速率的計算
11. 種系發生分析操作及運用步驟的流程圖
12. Some key points for performing a phylogenetic analysis
13. Methods for phylogenetic analysis
14. 世界個各地 EV71 的流行及其特性
15. 10 株 EV71 根據其 VP4 全長及 5''-NCR, VP1 部分核□酸序列之基因分型比較
16. 根據 5''-NCR 核□酸 99-748 序列自基因庫搜尋來自世界之 EV71 病毒株之種系分析圖
17. 根據 5''-NCR 核□酸序列 2442-3331 自基因庫搜尋來自臺灣之 EV71 病毒株之種系分析圖
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