石斑魚神經壞死症病毒去活化疫苗的製備及效能評估

Abstract

在台灣，病毒性神經壞死症（Viral nervous necrosis disease, VNN disease）每年均造成養殖業重大經濟損失，神經壞死症病毒主要感染石斑魚苗及幼魚，並引起高達80%~100%的死亡率。本研究主要為發展NNV去活化疫苗，並以浸泡法免疫魚苗，達到對抗NNV感染之目的。將NNV感染GF-1細胞，收集出現完整CPE後的病毒液，用於製備去活化疫苗及攻毒實驗。以相對存活率（Relative Percentage Survival, RPS）來評估疫苗的保護效果，免疫魚隻攻毒後RPS值至少要大於30%才具有顯著效果。本實驗先以四組不同的去活化疫苗（F-I,F-II,BPL,BEI）免疫石斑魚苗，結果顯示F-I,F-II和BEI組皆有顯著的保護效果，但BPL卻無。四組疫苗中，以BEI所製備的疫苗保護效果最好，RPS值高達83%~95%。其次，比較奈米包埋後的疫苗對免疫效果的影響，將F-II疫苗經奈米包埋後，以105 TICD50/ml病毒劑量免疫魚苗，攻毒後RPS值高達83%，保護效果最好，其RPS值甚至高於100倍病毒劑量的F-II未包埋疫苗組。比較不同浸泡濃度及浸泡時間對疫苗保護效果的影響，結果BEI疫苗的最低有效劑量為106 TICD50/ml。浸泡免疫20分鐘即可引發良好的保護效果。由浸泡攻毒實驗結果顯示，浸泡免疫一次BEI疫苗，保護效果至少可維持3個月。因此，使用NNV去活化疫苗浸泡免疫石斑八分苗，將成為重要且有效的防疫方法。

Nervous necrosis virus (NNV) is the most economically important pathogen of cultured groupers in Taiwan causing mass mortality 80~100% in fry and larvae. The aim of this study is to develop inactivated NNV vaccine, and a method of treating or preventing VNN through immersion immunization of grouper fry. The culture supernatant of NNV-infected GF-1 cells was collected after cytopathic effect (CPE) complete, and used for the preparation of inactivated vaccine and for challenge test. The effectiveness of vaccine was expressed by relative percentage survival (RPS), and significant protection usually means a RPS for vaccinated fish of at least above 30%. Four sorts of inactivated NNV vaccines (F-I, F-II, BPL, BEI) were prepared and used for immersion immunization of grouper fry. The result revealed that the inactivated vaccines (F-I, F-Il, BEI) could protect grouper fry from NNV infection, but BPL-prepared NNV vaccine failed to provide protection. Particularly, the RPS of BEI group was very high (83%~95%), and no virus was detected in vaccinated fish. Subsequently, the comparative results of vaccine without and with nano-encapsulation showed that effectiveness of nano-encapsulated vaccine improved. The RPS of fish immunized by encapsulated F-II vaccine was 83%, much higher than that of un-capsulated F-II vaccine with 100-fold viral concentration. The minimal NNV concentration for effective protection of BEI un-capsulated vaccine was 106 TICD50/ml. In addition, it was found that 20 min was enough for immersion immunization, but one month was necessary for the development of efficacious immunity after vaccination. Finally, the result of immersion challenge indicated that the protection of the vaccination could last for at least three months (RPS=82%). Consequently, NNV inactivated vaccine is believed to become an important approach for the control of NNV disease.