錦鯉疱疹病毒分離技術之建立及高敏感性診斷技術之開發

Abstract

錦鯉疱疹病毒 (koi herpesvirus, KHV)，目前被分類為Alloherpesviridae下的鯉科疱疹病毒第三型 (Cyprinid herpesvieus 3, CyHV3)，是一種會感染錦鯉 (Cyprinus carpio koi) 及鯉魚 (Cyprinus carpio carpio) 並造成高達80 % - 100 % 死亡率的高傳染性疾病。KHV於1998年最初發生於以色列，我國則於2002年12月出現首例。由於目前台灣地區尚無開發合適之細胞可供KHV病毒分離使用，使得本地KHV病毒之研究停滯不前。在許多研究報告中指出，由於環境溫度的改變及魚體免疫狀況的差異，推論KHV有潛伏感染的可能性，但目前仍缺少敏感性高且價廉之診斷技術可供鑑別健康魚隻及潛伏感染魚隻。 本實驗利用病魚乳劑接種待測細胞株，觀察細胞病變(Cytopathic effect, CPE) 並利用聚合酶鏈鎖反應 (Polymerase chain reaction, PCR)偵測病毒核酸。目前唯有本實驗室建立之錦鯉鰭細胞 (TKF1 cell) 可成功分離出台灣地區KHV。其他的錦鯉來源細胞株、金魚來源細胞株、肥頭鰷魚來源細胞株、非鯉科的石斑魚、鰻魚及吳郭魚來源細胞株及爬蟲類的甲魚來源細胞株，對KHV均不具有感受性。此外，我們利用間接免疫螢光染色技術發現細胞在感染KHV 12小時以上時，就能夠觀察到ORF 81蛋白質的陽性訊號。 本實驗並成功的建立了一套巢式聚合酶鏈鎖反應 (Nested polymerase chain reaction, nested PCR)，其特異性良好且在樣品中僅需含有17個病毒顆粒即可被偵測出來。目前已嚐試應用於一疑似潛伏感染場，配合該場病歷及nested PCR結果，認為此套高敏感性診斷方法具有應用於潛伏感染偵測之潛力。

Koi herpesvirus (KHV) was categorized as a member of the Alloherpesviridae under the species name Cyprinid herpesvirus 3 (CyHV3). It causes a highly contagious disease which leads up to 80 – 100 % mortality in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio). KHV was found firstly in 1998, and the first case in Taiwan has been identified in December 2002. Util now, there has been no suitable cells developed for KHV virus isolation in Taiwan, which becomes an obstacle for the research. Many previous researchers suggest the possibility in latent infection of KHV; however, we lack a diagnosis method providing higher sensitivity to distinguish the healthy and the latent infected fish. In our study, the KHV isolation, we inoculated the tissue homogenates of infected fish to tested cells, observed the cytopathic effect, and then detected the KHV DNA by polymerase chain reaction. Our results revealed that only Taiwan koi fin (TKF1) cell, established by our laboratory, showed the CPE and propagatde KHV successfully. Other cells derived from koi, goldfish, fathead minnow, grouper, eel, tilapia and soft-shell turtle were not susceptible for the KHV. Moreover, we detected the ORF 81 protein of KHV by indirect immunoflourescene assay, the positive signal appeared after 12 hours post infection. For the more sensitive diagnostic method, we developed a nested polymerase chain reaction with good specificity, and it was able to detect as less as 17 virions in the sample. We have been applied the diagnosis method to detect the fish from one koi farm, which was suspected to be the KHV endemic farm. According to the history of this farm and the results of nested PCR, we suggest the high sensitivity diagnosis method has the potential to apply to detect latent infection cases.