神經壞死病毒 (DGNNV) 感染的石斑魚幼苗死亡率接近100%。將此病毒所感染之石斑魚的組織切片，以蘇木紫與伊紅染色後，其視網膜的感光細胞層、外核層和內核層及中腦的視蓋可觀察到圓形空泡。以DGNNV之似病毒顆粒製備的抗血清進行間接螢光免疫偵測，可在石斑視網膜的內核層與中腦的視蓋發現病毒訊號。將DGNNV以眼球注射法感染斑馬魚，並觀察在96小時的神經組織變化，經H&E染色後，在斑馬魚視網膜的感光細胞層與中腦的視蓋組織觀察到空泡，而以間接螢光免疫偵測可於視網膜的外核層和中腦的視蓋發現病毒。本研究以重組基因法製備的似病毒顆粒來產生抗體，可用於偵測魚神經組織中的DGNNV。

The mortality of grouper larvae and juveniles infected by nervous necrosis virus (DGNNV) was near 100%. Vacuoles were found in photoreceptor layer, outer nuclear layer and inner nuclear layer of retina and optic tectum of mesencephalon for the dragon grouper that was infected by DGNNV, after stained with haematoxylin and eosin (H&E). Recombinantly expressed in E. coli, virus-like particles (VLPs) were used for antibody preparation. By indirect fluorescence antibody test (IFAT) with the mouse anti-VLP serum, DGNNV was detected in retina inner nuclear layer and mesencephalon optic tectum. At 96 hours post infection of DGNNV with intraocular injection, vacuoles were observed, with H&E staining, in zebrafish retina photoreceptor layer and mesencephalon optic tectum. In IFAT test, DGNNV was also detected in outer nuclear layer and optic tectum of zebrafish. This study showed antibody stimulated by the recombinant VLPs was sutible for DGNNV detection in fish nervous tissues.

摘要....................................................................................................................... I
ABSTRACT…………………………………………………………………….. II
目錄....................................................................................................................... III
圖目錄................................................................................................................... V
壹、前言............................................................................................................... 1
一、台灣石斑養殖現況................................................................................. 1
二、魚類神經壞死病毒 (Piscine nervous necrosis virus)............................. 2
三、病毒性神經壞死症 (viral nervous necrosis) 之病灶............................ 6
四、Alphanodavirus之基因........................................................................... 7
五、Betanodavirus之基因............................................................................. 12
六、似病毒顆粒 (virus-like particles, VLPs).................................................. 16
七、Betanodavirus的檢測法......................................................................... 22
八、利用細胞株偵測Betanodavirus............................................................. 23
九、RT-PCR偵測病魚神經組織................................................................... 28
十、利用電子顯微鏡偵測Betanodavirus..................................................... 29
十一、神經組織病理觀察............................................................................. 29
十二、利用抗體進行ELISA、IFAT或IHC偵測Betanodavirus............... 30
十三、研究目的............................................................................................. 32
貳、材料方法....................................................................................................... 33
一、斑馬魚及石斑魚樣品來源..................................................................... 33
二、複式顯微鏡與螢光顯微鏡..................................................................... 33
三、Lowry Method蛋白質定量..................................................................... 34
四、SDS-PAGE膠體電泳.............................................................................. 34
五、SSN-1細胞繼代...................................................................................... 35
六、龍膽石斑神經壞死病毒 (DGNNV) 繼代............................................ 36
七、病毒萃取................................................................................................. 37
八、病毒純化................................................................................................. 37
九、似病毒顆粒 (virus-like particles, VLPs) 製備與純化............................ 39
十、抗VLP之小鼠血清製備........................................................................ 41
十一、墨點法 (Dot blot) 檢測小白鼠產生抗體的力價............................. 42
十二、西方墨點法 (Western blot) ................................................................ 43
十三、DGNNV病毒感染.............................................................................. 44
十四、組織固定、滲蠟、包埋與切片......................................................... 44
十五、蘇木紫與伊紅染色............................................................................. 45
十六、細胞免疫螢光偵測............................................................................. 46
十七、組織免疫螢光偵測............................................................................. 47
十八、EZ-C1軟體操作.................................................................................. 48
十九、ImageJ軟體去除自體螢光的方法..................................................... 49
參、結果............................................................................................................... 51
一、神經壞死病毒 (DGNNV) 感染SSN-1細胞........................................ 51
二、DGNNV病毒與似病毒顆粒之結構相似性.......................................... 52
三、石斑魚與斑馬魚的腦與視網膜組織切片............................................. 53
四、DGNNV所感染的病魚其視網膜與視蓋組織之圓形空泡.................. 54
五、影像處理以去除自體螢光及背景......................................................... 55
六、anti-VLP抗體偵測DGNNV感染之病魚的眼和腦組織..................... 57
肆、討論............................................................................................................... 58
伍、參考文獻....................................................................................................... 64
陸、圖表............................................................................................................... 88
附錄A、魚的視網膜與腦部構造....................................................................... 105
附錄B、藥品配製................................................................................................ 108

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