錦鯉疱疹病毒在持續感染錦鯉中排毒及潛伏位置之探討

Abstract

錦鯉疱疹病毒 (koi herpesvirus, KHV) 為二十面體，病毒蛋白鞘直徑約為110 nm，具有封套的雙股DNA病毒，核酸大小為295 kbp，被分類為鯉魚疱疹病毒第三型 (Cyprinid herpes virus - 3, CyHV - 3)，2009年被歸類為Alloherpes- viridae。本病最初在1997年爆發於德國，美國及以色列則在1998年爆發疫情，經過國際間的貿易及運輸，此病藉此傳播開來，世界各國陸續有疫情傳出，而在2002年12月，台灣已確定有錦鯉感染本症。本病目前尚未了解病毒如何存在於環境中，推測病毒或許能夠保存在糞便、底泥或宿主細胞內，待氣溫回暖時再度引起魚隻的感染。 一般疱疹病毒感染後，病毒會在宿主體內行潛伏感染及病毒再活化，而其他魚類的疱疹病毒，如Cyprinid herpesvirus 1 (CyHV-1)、Channel catfish virus (Ictalurid herpesvirus 1) 及Eel herpesvirus (Herpesvirus anguillae)，感染魚隻後，已被證實有此二特性；另外，許多曾發生過KHV疫情之錦鯉養殖場，在春、秋兩季，常又有因KHV感染而發病死亡之病例。故本研究之目的為探討KHV感染魚隻後，是否會形成帶原者 (carrier) 與KHV行潛伏感染時在魚體內的潛伏位置及病魚的排毒模式。本研究利用水溫的變化，使KHV感染的魚隻形成carrier後，並利用本實驗室所建立之特異性良好且敏感性較高的巢式聚合酶鏈鎖反應 (Nested polymerase chain reaction, Nested PCR)，檢測KHV在魚體內的潛伏位置。其結果顯示KHV帶原魚不論在32oC或23oC皆會排毒而感染健康魚隻，且在KHV行潛伏感染的魚隻，以Nested PCR檢測鰓、肝、脾、腎、前腎、腦、腸、生殖腺等臟器皆為陽性反應，故推論KHV在潛伏感染時，並無特定潛伏臟器。然而PCR檢測結果多為陰性，故在往後在防疫上，須以Nested PCR檢測KHV潛伏感染魚隻，以避免出現偽陰性之情況。

Koi herpesvirus (KHV) is an enveloped virus with an icosahedral electron-dense core of 100-110 nm surrounded by tegument protein. The genome of the KHV comprises linear double-stranded DNA (dsDNA) of 295 kbp and its genomic sequence was similar with Cyprinid herpesvirus 1 and 2, so was named as Cyprinid herpesvirus 3. In 2009, KHV was classified as Alloherpesviridae. The first outbreak of this disease was reported in 1997 in Germany, then USA and Israel in 1998. Rapid spread of KHV occurred because of the trade of koi, and in December 2002, KHV has been identified in Taiwan. How and where KHV preserve in the environment and in carrier fish is important, but still unknown. Many researchers proposed that the virus may persist in fish droppings, sediments, and survived fish after infection, or preserve in other fish species and animals, then when the water temperature is to a permissive temperature, virus will be released and infect carp again. Generally herpesviruses including fish herpesvirus show characters about latent infection and reactivation. In spring and autumn, many koi farms which had been diagnosed KHV positive, are often with fatal reports due to the recurrence of KHV in Taiwan. The aim of this study is to investigate whether KHV survived fish will become carriers, and which organs or tissues will KHV be latent, and whether the virus be released from KHV carrier, if the water temperature was proper. In this study, we changed water temperature to induce KHV carriers and these carrier would cohabitate with naïve fish, and then, locate the KHV by a more sensitive and specific diagnostic method, nested polymerase chain reaction (nested-PCR). Our results indicated that KHV carrier can transmit KHV to naïve fish no matter in 23oC or 32oC and that KHV TK gene can be detected by nested PCR in all organs (gill, liver, spleen, kidney, head kidney, brain, intestine and gonad) in carrier fish and cohabitated fish. Thus, our results suggest that KHV exist with very low level in internal organs without favorite during persistent infection. We also suggest that PCR is not the accurate method to screening carrier, and need the more sensitive method to detect the carrier fish.