評估台灣錦鯉疱疹病毒福馬林去活化疫苗引起之免疫效果與保護效力

Abstract

錦鯉疱疹病毒 (Koi herpesvirus，KHV)，亦稱為鯉魚疱疹病毒第三型 (Cyprinid herpes virus-3， CyHV-3)，或鯉魚間質性腎炎及鰓壞死病毒 (carp interstitial nephritis and gill necrosis virus，CNGV)，為一具封套的二十面體病毒，歸屬於 Alloherpesviridae科。KHV自1998年於以色列爆發以來，2000年便已在世界各地接連傳出災情，且由世界動物衛生組織（World Organization for Animal Health，OIE）列為水生動物輸出入檢疫疾病。我國亦於2002年12月出現首項確診病例。目前台灣的錦鯉養殖場在季節交替之際，時常傳出KHV感染症的病情報告，但截至目前為止，除了隔離病魚、撲殺與清池外，尚無有效防治手段。因此KHV之疫苗開發刻不容緩。 2003年Ronen等人藉由KHV的連續繼代方式製造減毒疫苗，但由於臨床上無法免除病毒毒力回復之風險，因此限於以色列地區使用。本實驗著眼於死毒疫苗的製作，透過福馬林去活化疫苗的接種，引發錦鯉魚隻的體液免疫反應，並配合弗氏完全佐劑（Freund’s complete adjuvant，FCA）與Montanide ISA 763A兩種佐劑，以增強魚隻的細胞免疫反應，旨在建立魚隻抵抗KHV感染的能力。結果顯示，在Naive魚隻上，未配合佐劑的死毒疫苗可引起高達1:128的血清中和抗體力價，相對存活率（relative percent survival，RPS）亦超過70%；而在Carrier上的RPS甚至可達100％。配合佐劑的疫苗，其血清表現中和抗體力價的時間，較未配合佐劑的超出一倍，在免疫後96天仍可偵測到1:32以上的中和抗體力價。 本實驗成功地在實驗室環境下，透過活體in vivo試驗，確認了KHV福馬林去活化疫苗的效果，未來將進一步將本死毒疫苗之製造條件予以最佳化，以求得更低的成本與更高的保護效率。

Koi Herpesvirus (KHV) which is also called Cyprinid herpes virus-3, (CyHV-3) or carp interstitial nephritis and gill necrosis virus (CNGV) is a icosahedral virus and grouped family Alloherpesviridae. The disease firstly reported in Israel in 1998, then outbreaked around the world until 2000. The huge economical losing has lead KHV to be recognized as certificated disease by World Organization for Animal Health (OIE). In Taiwan, the first case report has been found in December in 2002. The infection of KHV usually took place during season changing in Koi aquaculture field, and there are still no effective control strategies for KHV infection except quarenting, disinfection and pond liming. Therefore the establishment of vaccine is an urgent task to pursue. Ronen et. al. have developed an attenuated KHV strain as a vaccine through series passaging methods. But the using of attenuated vaccine is totally restricted in Israel area due to the significant virulence recovery risks. This research was mentioned about the Formalin-inactivated method to produce high effective vaccine for inducing humeral immunity of fishes. On the other hand, we also select two adjuvants including Freund’s complete adjuvant (FCA) and Montanide ISA 763A to build up the cell-mediated immunity while vaccing inoculating. After immunizing naive fishes, the high titer serum neutralizing antibodies can be detected, and the relative percent survivol (RPS) was over 70%. And the RPS on Carriers was even approaching 100%. The neutralizing antibodies lasting of inactivated vaccine with adjuvant are two times longer than the vaccine without adjuvant. The titer of serum antibody still reached 1:32 at 96th day post-immunization. We successfully established an effective inactivated KHV vaccine in vivo. We will optimize the producing contidion in order to reduce the cost and raise the efficacy.