自不同魚種分離之神經壞死症病毒株病原性的比較

• Main Authors: 廖睿雯, Liao, Rui-Wen
• Other Authors: 齊肖琪, 臺灣大學：動物學研究研究所
• Format: Thesis
• Language: Chinese; English
• Published: 2004
• Subjects: 魚類神經壞死症病毒; 石斑魚; Grouper; nodavirus; Anguilla anguilla; European eel
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/56647; http://ntur.lib.ntu.edu.tw/bitstream/246246/56647/1/ntu-93-R91225033-1.pdf

神經壞死症病毒(Nervous Necrosis Virus, NNV)在世界各地已引起三十幾種以上海水養殖魚苗大量死亡。本實驗在探討自不同魚種分離的NNV分離株對石斑魚的病原性有何差異。本實驗將分離自石斑（Epinephelus coioides）、歐洲鰻（European eels , Anguilla anguilla）、黃鱲鰺（yellow-wax pompano, Trachinotus falcatus）、金目鱸（baramundi, Lates calcarifer）、海鱺（cobia, Rachycentron canadum）、老鼠斑（Cromileptes altivelis）及中國鯰（Chinese catfish , Parasilurus asotus）等7個台灣分離株及分離自北歐冷水域的大西洋比目魚（Atlantic halibut, Hippoglossus hippoglossus）分離株，對點帶石斑幼魚進行攻毒實驗，僅三株台灣NNV分離株石斑幼魚死亡。進一步便以的海水養殖老鼠斑的分離株(HGNNV)、淡水養殖中國鲶魚的分離株(CCNNV)，以及來自冷水魚的大西洋比目魚分離株 (AHNNV)對點帶石斑苗進行攻毒，結果HGNNV引起的死亡率是72%，CCNNV是29%，而AHNNV只有1%。經由RT-PCR檢測，死亡魚體內都測得到用來攻毒的NNV病毒株，但AHNNV的含量很低， 所以AHNNV可感染得上石斑魚，只是病毒複製量非常低，經由AHNNV感染GF-1細胞後再不同溫度培養的實驗結果，推測與病毒核酸複製最適溫度的差異有關。瀕死魚的腦及視網膜有嚴重空泡化現象，殘活魚雖無此現象，但測得到病毒；免疫組織染色發現，殘活幼魚視網膜附近的黑色素巨噬細胞中心有NNV蛋白質存在，推測殘活魚低病毒量帶原與黑色素巨噬細胞非專一性的免疫功能有關。 Nervous necrosis virus (NNV) has caused mass mortality in more than 34 fish species. The aim of this study is to compare the pathogenecity of NNV isolated from different species of fish. Seven Taiwan NNV isolates and one European NNV isolate were used for the challenge of grouper juveniles. The clinical syndromes observed in all the fish challenged with the seven Taiwan NNV isolates, but not in the fish challenged with Atlantic halibut NNV (AHNNV). The mortality occurred only in the fish challenged with Baramundi NNV (BNNV), European eel NNV (ENNV) and hump-back grouper NNV (HGNNV). In advance, grouper larvae reared at 25°C were bath-challenged with HGNNV isolated from warm water marine fish, Chinese catfish NNV (CCNNV) isolated from fresh water fish and AHNNV isolated from cold water fish. The accumulated mortality of HGNNV was 72%, CCNNV was 29%, and AHNNV was 1%. The moribund grouper larvae were examined by RT-PCR and semi-nested PCR, and NNV RNA was found in the brain and retina of the fish. Hence, AHNNV was able to infect grouper larvae, but the level of AHNNV RNA in the challenged grouper was very limited. The temperature effects on the replication of AHNNV were tested in GF-1 cells. The RNA of AHNNV could be detected in the infected GF-1 cells incubated at 20°C but not at 28°C. It is therefore suggested that the low level of AHNNV in the challenged grouper is due to the unproper temperature (28°C) for AHNNV RNA-dependent RNA polymerase (RdRp). Vacuolation of brain and retina was only observed in the moribund fish but not in the fish survived viral nervous necrosis disease. Virus was revealed in the brain and retina of survivor fish by immunohistochemistry (IHC) staining and immunofluorescent (IF) staining. In particular, it is the first time to find NNV in the melanomacropahge center (MMC) within the retina of survivor fish. 致謝……………………………………………………………………….I 目錄………………………………………………………………………II 圖表目次……………………………………………………………….III 中文摘要…………………………………………………………………IV 英文摘要………………………………………………………………….V第一章 前言…………………………………………………………….1 第二章 文獻回顧…………………………………………………………2 1. 病毒性神經壞死症病毒之病史及臨床症狀…….……….2 2. VNN病理及組織分佈研究……………………………………3 3. 病毒外觀、特性及化學敏感性…………………………….3 4. 病毒診斷…………………………………………………….4 5. 病毒的分類地位…………………………………………….5 6. 地理分佈與宿主範圍……………………………………….5 7. 神經壞死症病毒的流行病學……………………………….6 第三章 材料與方法…………………………………………………….8 1. 攻毒試驗…………………………………………………….8 2. 病毒核醣核苷酸的萃取與偵測……………………………9 3. 免疫組織化學染色…………………………………………11 4. 免疫螢光染色………………………………………………13 5. 中和反應試驗………………………………………………14 第四章 結果……………………………………………………………15 1. 8株NNV分離株對體長13 cm石斑幼魚病原性的比較…….15 2. 三株NNV分離株對體長3 cm石斑苗病原性的比較……….16 3. 不同溫度對三株病毒複製效率的影響……………………17 第五章 討論……………………………………………………………19 第六章 參考文獻………………………………………………………24 圖 表 目 次 圖一､不同NNV分離株攻毒九天後的病癥比較………………………30 圖二､免疫組織化學染色…………………………………………….31 圖三､免疫組織化學染色與螢光染色結果………………………….32 圖四､三種NNV分離株攻毒後累計死亡率的比較……………………33 圖五､三種NNV分離株攻毒後死亡及無病癥魚PCR檢測結果……….34 圖六､免疫組織化學染色…………………………………………….35圖七､三種NNV分離株不同溫度培養下PCR檢測結果……………….36 表一､攻毒實驗所使用的病毒株資料……………………………….37 表二､點帶石斑魚苗(13 cm)肌肉注射攻毒採樣分析結果…………38 表三､病毒於室溫吸附然後培養6日之力價…………………………39 表四､病毒個別在20°C及28°C吸附及複製效應比較……………….40