張繼堯Chang, Chi-Yao臺灣大學:漁業科學研究所楊雁南Yang, Yen-NanYen-NanYang2010-05-102018-07-062010-05-102018-07-062009U0001-1808200915522200http://ntur.lib.ntu.edu.tw//handle/246246/181717神經壞死病毒及虹彩病毒是台灣水產養殖重要的感染病原，近年來鄰近各國也相繼傳出疫情，台灣雖有養殖魚類監控報告，但是甚少對沿近海域野生魚類進行神經壞死病毒及虹彩病毒的檢測分析。本實驗分別以RT-PCR和PCR方法檢測神經壞死病毒及虹彩病毒，再進一步用Nested-PCR方法提高檢測靈敏度，藉此了解台灣沿近海域野生及養殖魚類帶原病毒情況。195個樣本中有48個在神經壞死病毒Nested-PCR檢出、32個在虹彩病毒Ranavirus Nested-PCR檢出、在虹彩病毒Megalocytivirus檢測中則沒有檢體被檢出，此結果顯示台灣沿近海域的魚類帶原神經壞死病毒情形普遍(檢出率高達25.6%)，Ranavirus也有不少帶原情形(檢出率16.4%)，另外有7個樣本同時檢出神經壞死病毒及Ranavirus帶原。雙重帶原的樣本中6個來自宜蘭大溪漁港，1個來自墾丁南灣採集的野生魚類，顯示野生魚類同時帶原兩種病毒在自然界中是存在的。所檢測出神經壞死病毒檢體外套蛋白基因序列進行病毒基因比對後，發現38個神經壞死病毒檢體中有34個屬於RGNNV(佔89.4%)、4個屬於SJNNV(佔10%)，顯示台灣海域魚類帶原神經壞死病毒以RGNNV基因型為主；虹彩病毒方面在檢出Ranavirus帶原檢體中，全數33個檢體其主要外鞘蛋白基因序列皆和石斑魚虹彩病毒(Grouper Iridovirus; GIV)及新加坡石斑魚虹彩病毒(Singarpore Grouper Iridovirus; SGIV)相似度高達98-99%，但和同為Ranavirus屬的蛙虹彩病毒(Bohle iridovirus; BIV)、流行性造血組織壞死症病毒( Epizootic haematopoietic necrosis virus; EHNV )相似度只有68-70%；澎湖箱網養殖的棕點石斑檢出虹彩病毒Megalocytivirus，比對其CY15 amplicon基因序列和1992年自高雄養殖石斑魚檢出的台灣石斑虹彩病毒(Grouper Iridovirus of Taiwan; TGIV)相似度為90%，比和福建福州養殖大黃魚檢出的虹彩病毒(Large Yellow Croaker Iridovirus; LYCIV )的99%相似度還低，顯示除原先的TGIV以外在澎湖地區尚有其他Megalocytivirus存在。Betanodavirus and iridovirus are the major viral pathogens that seriously damage aquaculture industry in Taiwan and other Asian countries. Although virus surveillance has been performing for cultured fish in Taiwan, however, there are few reports of the detection of both viruses in coastal fish. In this study, molecular detection methods such as PT-PCR and PCR were used to detect betanodavirus and iridovirus. To detect trace amount of virus in the healthy carrier fish, the Nested-PCR method was further introduced to increase the detection sensitivity. In total 195 wild fish samples, 50 samples were positive in NNV Nested-PCR (detection rate 25.6%), 32 samples were positive in Ranavirus Nested-PCR (detection rate 16.4%), and no sample was positive in Megalocytivirus Nested-PCR. These data indicate that wild fish were NNV and Ranavirus carriers around Taiwan seashore in general. Seven fish (6 from Yi-Lan, 1 from Keenting) were positive both in NNV and Ranavirus Nested-PCR. Therefore, it is possible that one individual fish can be simultaneously infected by two viruses in nature. The phylogenic tree shows that 34 of 38 NNV samples belong to RGNNV group (89.4%), whereas 4 belong to SJNNV group (10%). All 33 of Ranavirus samples belong to GIV and SGIV group, but not BIV (Bohle iridovirus) nor EHNV (Epizootic haematopoietic necrosis virus) group. Both phylogenic tree and sequence similarity of Megalocytivirus (CY15 amplicon gene) from Peng-Hu cultured Tiger grouper (Epinephelus fuscoguttatus) are closer to LYCIV (Large Yellow Croaker Iridovirus) in Fujian but not to TGIV(Grouper Iridovirus of Taiwan).中文摘要……………………………………...................................................................I文摘要……………………………………....................…….....................................Ⅲ錄…………………………………………................................................................ IV目次……………………………………….......................……..………………….. VII目次……………………………………………….……………....................……....Ⅷ、 文獻整理…………………..………………….…….....…….............…..………1.1 魚類神經壞死病毒簡介…..….............................................…….........……1.2 魚類虹彩病毒簡介…………………………………………....................... 7.3 台灣魚類感染神經壞死病毒及虹彩病毒近況.……………………….…11、 材料與方法………………………………………..………..................……….14.1魚類檢體採集………..……………….………………....................…….…14 2.1.1 野生魚類檢體來源……………………………….......................…14 2.1.2 養殖魚類檢體來源……………………………………...............…14 2.1.3 檢體處理歸檔…………………………………….......................…14.2 魚類神經壞死病毒與虹彩病毒分子檢測與特定基因序列分析比較…..14 2.2.1 RNA及DNA之萃取…………………………………………..….…16 2.2.2 RT-PCR、PCR及Nested PCR………..….…..….................……….17 2.2.3 定序及病毒特定基因序列分析……….......................................…18.3 檢體病毒液感染特定細胞株細胞病理觀察…………................………..19 2.3.1 病毒液製備……………………………………….......................…20 2.3.2 檢體病毒液感染特定細胞株細胞病理觀察................................ ...21、 結果………………………………………………………….......................…..22.1 檢體分類及鑑定整理..................................................................................22.2 魚類神經壞死病毒與虹彩病毒分子檢測結果.………................……….22.3 魚類病毒與檢體特定基因序列分析及比較…………………............…..23.4 檢體病毒液感染特定細胞株細胞病理觀察……..………..................…..23、 討論…………………………………………………………...................……..25.1 台灣沿近海野生魚類帶原神經壞死病毒情況..........................................25.2 台灣魚類感染虹彩病毒及帶原情況……..................................................27.3 同一檢體檢出神經壞死病毒及虹彩病毒..................................................30、 參考文獻……………………………………………………................……….32、 圖……………………………………………………………................……….39、 表……………………………………………………………................……….46、 附錄…………………………………………………………................……….52application/pdf1137723 bytesapplication/pdfen-US分子檢測虹彩病毒神經壞死病毒野生魚類疾病監測detectionnodavirusiridivirusMegalocytivirusNNVGIVRanavirushttp://ntur.lib.ntu.edu.tw/bitstream/246246/181717/1/ntu-98-R96b45010-1.pdf