Developments of an Isolation System and a Nested PCR Diagnosis Method with Superior Sensitivity for Koi Herpesvirus

建立錦鯉疱疹病毒分離細胞株及高敏感度診斷技術之開發

李建穀(Jiann-Gwu Lee) ； 劉馨淳(Shin-Chun Liu) ； 陳媺玫(Meei-Mei Chen)

《臺灣獸醫學雜誌》 40卷1期 (2014/03) Pp. 15-25

摘要

錦鯉疱疹病毒（koi herpesvirus，KHV）為一具高傳染性之魚類疾病，可感染錦鯉（Cyprinus carpio koi）及鯉魚（Cyprinus carpio carpio），並造成高達80－100%之死亡率，以及重大經濟損失。由於環境溫度改變能影響魚體免疫狀態，造成KHV無臨床症狀之潛伏感染，若無高敏感性之診斷技術以供篩檢，將無法證明KHV帶原者之存在。本實驗室首次建立之錦鯉鰭細胞（TKF-1 cell）培養，可成功提供台灣株KHV之分離及增殖；而其他國內外之細胞培養，如錦鯉來源細胞株、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等細胞株，對KHV均不具有感受性。同時本實驗成功地建立一套巢式聚合酶鏈鎖反應（nested PCR）技術，其特異性良好且敏感度極高，在樣品中僅需含有17個病毒顆粒即可被偵測出來。此技術亦為本實驗室證實，能於疑似KHV潛伏感染魚場中之健康魚隻體中檢測出KHV。TKF-1細胞株之建立以及nested PCR技術之開發，對台灣特有KHV之診斷，尤其是潛伏感染魚隻，具有高度偵測之潛力，並可提供早期防治之對策。

關鍵字

錦鯉 ； 疱疹病毒 ； 魚鰭細胞 ； 巢式聚合酶連鎖反應 ； 台灣

並列摘要

Koi herpes virus (KHV) infection is an emerging, highly contagious fish disease that has imposed high mortality in common carp and koi. Previous studies have shown that the difficulty in viral identification might be due to the latent infection of KHV in Koi. In the present study, we reported the establishment of methodologies for KHV diagnosis. Taiwan koi fin (TKF-1) cells were found to be the only type of cell culture that KHV could be sustained and the cytopathogenic effects (CPE) could be induced directly by the inoculation of the homogenates of KHV-infected fishes tissues. Other cells cultures derived from koi, goldfish, fathead minnow, grouper, eel, tilapia and soft-shell turtle were proven not to be susceptible for the KHV. KHV infection could be further confirmed by the detection of the 409 bp fragments of KHV DNA using polymerase chain reaction in TKF-1 cells. Moreover, level of ORF 81 protein of KHV could be determined by indirect immunofluorescence assay 12 hours post infection. In addition, to increase the specificity and sensitivity in diagnosing KHV, we developed a nested PCR protocol capable of detecting as few as 17 KHV virions. This technology was verified with the suspected but asymptomatic koi and was proved to be a more sensitive tool for diagnosing KHV-infected koi during latent infection than others. This is the first report in Taiwan regarding the establishment of koi cell cultures for the propagations of locally isolated KHV and the development of a new diagnostic method with higher sensitivity for KHV infection.

並列關鍵字

carp ； koi herpesvirus ； koi fin cell ； nested-PCR ； Taiwan