錦鯉疱疹病毒潛伏感染機制之探討

Study of Koi Herpesvirus Persistent Infection Mechanism

杜牧融(Mu-Jung Tu)

指導教授 ： 陳媺玫

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01590

摘要

錦鯉疱疹病毒 (Koi herpesvirus, KHV ) 的爆發造成養殖業者的極大損失，目前KHV被分類為Alloherpesviridae 下的鯉魚疱疹病毒第三型 (Cyprinid herpesvirus 3, CyHV-3)，具封套、雙股DNA，核酸大小約為295kbp，對於初次感染的家鯉或錦鯉有極高的死亡率，耐過錦鯉疱疹病毒攻擊的鯉魚會成為帶原魚，而其持續感染的機制目前尚不清楚。潛伏感染是持續感染的一種方式，其為疱疹病毒科的特色。除了常見的哺乳類之疱疹病毒科 (Herpesviridae) 會潛伏感染外，Alloherpesviridae中的河鯰病毒 (Channel catfish virus)、鯉魚疱疹病毒第一型 (Cyprinid herpesvirus-1) 也證實會產生潛伏感染。而KHV可因為溫度變化而造成潛伏感染，其潛伏位置包含很多器官以及周邊血球。無症狀的帶原鯉魚溫度改變或遭受壓力時，疾病就會再度爆發。因此，本研究想了解錦鯉疱疹病毒持續感染的機制，根據文獻指出病毒持續感染的機制可能是由於病毒DNA插入宿主染色體、病毒產生環狀的游離基因 (episome)、細胞產生干擾素或是病毒產生與潛伏相關的轉錄 (LATs)。本實驗以帶原錦鯉進行初代細胞培養，建立兩株帶原KHV之持續性細胞株。從生長速度、染色體數目等細胞特性分析顯示其與健康細胞株有差異，且該細胞不接受水生動物來源之病毒重複感染。其釋出之病毒力價較低，攻毒實驗魚不造成死亡，用原位雜合法僅在實驗魚之鰓及腎偵測到陽性訊號。以此兩株細胞進行持續感染機制探討，首先比較帶原細胞株釋出之病毒與標準病毒的力價，以及於實驗魚體內分布狀況、帶原細胞株是否會分泌干擾素、帶原細胞的病毒有無形成環狀的游離基因體、或是病毒基因嵌入細胞染色體之可能。目前結果顯示帶原細胞株干擾素的分泌或病毒嵌入宿主染色體可能都是造成KHV於此二株細胞內持續感染的機制。

關鍵字

錦鯉疱疹病毒, 持續感染, 干擾素, 原位雜合, 病毒基因嵌入染色體

並列摘要

Koi herpesvirus (KHV) infection cause severe financial losses. KHV is a member of Alloherpesviridae, and newly designated species Cyprinid herpesvirus 3. The virion have an outer envelope and an icosahedral capsid contains a single, linear, double-stranded DNA of 295 kbp. Common carp (Cyprinus carpio carpio) and Koi carp(Cyprinus carpio koi) are the only species known to be affected by KHV. The survivors will become carrier fishes, but persistent infection mechanism of KHV is not clear. One of the unique features of Herpesviridae is latency which had been confirmed in Channel catfish virus and Cyprinid herpesvirus infection model. KHV can become latent in the peripheral white blood cells and various tissues, and will reactivation while temperature shifting, or been forced. The aim of this study is trying to find out persistent infection mechanism of KHV. Persistent infection mechanism in tissue culture includes interferon, intranuclear episome, viral DNA insertion, or virus producing latency-associated transcripts. We established two KHV persistently infected cell lines from carrier fishes through primary cell culture. Persistent infection cells characteristics such as growth rate and chromosome analysis are different from healthy cells. These two cells do not accept superinfection of virus from aquatic sources. The virus titer of persistently infected cells is fluctuation, and have no ability to induce fish mortality. The results of in situ hybridization showed the KHV positive signals in gill and kidney. In this study, virus produced by the two persistently infected cells are compared to the wild KHV, such as virus titer and organ distribution. Current results showed that the mechanism of KHV persistent infection is relate to interferon releasing and virus genome insertion.

並列關鍵字

Koi herpesvirus, persistent infection, interferon, in situ hybridization, viral genome insertion

參考文獻

劉馨淳, 2009, 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. 國立台灣大學獸醫學研究所碩士論文.

徐達, 涂堅, 鄭穹翔, 陳媺玫, 2007, 錦鯉疱疹病毒傳播媒介之探討. Taiwan Vet J 33: 88-95.

Adamek, M., Rakus, K. L., Chyb, J., Brogden, G., Huebner, A., Irnazarow, I., Steinhagen, D., 2012, Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections. Fish Shellfish Immunol 33: 482-493.

Aoki, T., Hirono, I., Kurokawa, K., Fukuda, H., Nahary, R., Eldar, A., Davison, A. J., Waltzek, T. B., Bercovier, H., Hedrick, R. P., 2007, Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide. J Virol 81: 5058-5065.

Arbuckle, J. H., Medveczky, M. M., Luka, J., Hadley, S. H., Luegmayr, A., Ablashi, D., Lund, T. C., Tolar, J., De Meirleir, K., Montoya, J. G., Komaroff, A. L., Ambros, P. F., Medveczky, P. G., 2010, The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro. PNAS 107: 5563-5568.