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研究生:莊蕙菁
研究生(外文):Hui-Ching Chuang
論文名稱:石斑魚神經壞死病毒特性及檢測方法之研究
論文名稱(外文):Characterization of Grouper Nervous Necrosis Virus and Development of Diagnosis Method.
指導教授:陳宗嶽
指導教授(外文):Tzong-Yueh Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:118
中文關鍵詞:病毒檢測神經壞死病毒即時螢光定量聚合酶連鎖反應石斑魚
外文關鍵詞:virus diagnosisgroupernervous necrosis virusreal-time PCR
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中文摘要
  病毒性神經壞死症(Viral nervous necrosis disease,VNN disease)為近年來臺灣石斑魚養殖上極為嚴重的疾病,其致病原為神經壞死病毒(Nervous Necrosis Virus),此病毒感染易造成石斑魚苗及幼魚大量死亡,死亡率往往高達100%,造成臺灣石斑養殖業極大的損失。該病毒帶有兩條單股RNA,分別為RNA1與RNA2,其中RNA1的功能為RNA-dependent RNA polymerase;RNA2則可轉譯出病毒的外殼蛋白質。
  本研究中,於2002年至2004年期間,至台南及高雄地區數個石斑魚養殖場中收集罹患病毒性神經壞死症之病魚,其中包含各種臺灣地區熱門養殖石斑魚種:點帶石斑、龍膽石斑以及金錢斑;利用RT-PCR選殖RNA1與RNA2基因序列;在RNA1部分一共選殖了9個分離株,由胺基酸序列比對後可區分成8種變異株,彼此相似度在97.2~99.9%之間。RNA2共選殖了13個分離株,在外殼蛋白的胺基酸序列比對後發現2種變異株,僅在第214個胺基酸上產生變異(T/N),相似度高達99.6%~100%之間,其分子演化歸屬於RG-NNV基因型。而RNA1中的5個變異株在石斑魚鰭細胞中複製能力無明顯差異。
  另一方面,利用SYBR Green I chemical,我們發展檢測NNV之螢光即時PCR(real-time PCR)系統,其偵測極限可達到190病毒copies,和傳統RT-PCR相比,其靈敏度高於十倍。藉由此系統進行NNV在罹病龍膽石斑魚組織分佈的定量,發現NNV存在於各器官中,但含量有顯著差異,其中以腦部及眼睛最多,以每公克組織而言,平均高於其他組織1000倍左右。而由三種石斑魚細胞株進行NNV in vitro 之複製實驗,結果顯示NNV在石斑魚鰭細胞中病毒複製生長為最佳,造成明顯細胞病變(cytopathic effect, CPE),以及嚴重導致細胞死亡,其複製產物高於肝細胞株及腎細胞株的100~1000倍。藉此我們推測神經壞死性病毒在複製生長上有組織侵性(tropism),且對細胞傷害能力有差異。
Abstract
 In recent years, viral nervous necrosis disease (VNN)was the greatest disease of hatchery-reared grouper in Taiwan . The causative agent of VNN was nervous necrosis virus (NNV)and the mortality of grouper larvae and juvenile during disease outbreak were usually above 90%. NNV contains two, single stranded, positive-sense RNA called RNA1 and RNA2.RNA1 encodes RNA dependent RNA polymerase, RNA2 encodes viral coat protein.
 In our study, we collected VNN diseased grouper samples from several aquaculture farms near Tainan from 2002 to 2004 which including orange-spotted grouper(Epinephelus coioides), giant grouper(Epinephelus lanceolatus) and potato grouper(Epinephelus tukula).We cloned RNA1 sequences from 9 isolates and RNA2 sequences from 13 isolates. Then using Clustal W program to compare the amino sequences. The result showed that according to the deduced amino acid of RNA1 gene, there were 8 variation strains out of 9 isolates and with an amino acid identity of 97.2% to 99.9% between these strains. On the other hand, according to the amino acid sequences of NNV capsid protein, there were 2 variation strains out of 13 isolates. The two strains with an amino acid identity of 99.6% to 100%, and share the same point mutation site (a.a. 214). All the 13 isolates belong to RGNNV genotype.
 We also developed a real-time quantitative PCR diagnosis system for NNV, the detectable limit was 190 virus copies, and the sensitivity was 10 times than conventional PCR detection. By using real-time quantitative PCR detection, we knew that NNV could be found in many organs, but the virus quantity in brain and eyes were usually 1000 times than other organs in Giant grouper. The virus propagation test in vitro showed that grouper fin cell line(GF-1)could propagate 100 to 1000 times virus particles than liver and kidney cell lines, these two results indicate that there were tissue tropism when virus infected cell and propagated. Finally, we chose 5 RNA1 variation strains to test their replication ability, but there was no significant difference between these strains.
目錄
中文摘要. . . . . . . . . . . . . . . . . . . . . . . . .Ⅰ
英文摘要. . . . . . . . . . . . . . . . . . . . . . . . .Ⅱ
目錄. . . . . . . . . . . . . . . . . . . . . . . . . . .Ⅲ
圖目錄. . . . . . . . . . . . . . . . . . . . . . . . . .Ⅳ
表目錄. . . . . . . . . . . . . . . . . . . . . . . . . .Ⅴ
第一章、文獻探討
第一節、石斑魚簡介與臺灣養殖現況. . . . . . . . . . 1
第二節、海水魚病毒神經性壞死症. . . . . . . . . . . 2
第三節、神經壞死病毒. . . . . . . . . . . . . . . . 6
第四節、實驗目的. . . . . . . . . . . . . . . . . . 13
第二章、材料與方法. . . . . . . . . . . . . . . . . . . 14
第三章、實驗結果
第一節、石斑魚神經壞死病毒之檢測與基因組序列分析. . .34
第二節、石斑魚神經壞死病毒之分離與純化. . . . . . . .36
第三節、即時螢光定量PCR之系統建立. . . . . . . . . . 37
第四節、石斑魚神經壞死病毒感染特性之研究. . . . . . .40
第四章、討論. . . . . . . . . . . . . . . . . . . . . . .44
第五章、參考文獻. . . . . . . . . . . . . . . . . . . . .51
圖表. . . . . . . . . . . . . . . . . . . . . . . . . . .57
附錄. . . . . . . . . . . . . . . . . . . . . . . . . . .99
自述. . . . . . . . . . . . . . . . . . . . . . . . . . .105
謝淨如(2002),魚類神經壞死症病毒單源抗體之建立定性及應用,國立臺灣大學動物學研究所碩士論文,台北。

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