神經壞死症病毒與石斑魚虹彩病毒雙價疫苗對石斑魚保護效力之研究

Abstract

石斑魚為亞洲地區水產養殖產業之重要經濟魚種。神經壞死症病毒 (Nervous necrosis virus, NNV)及石斑魚虹彩病毒(Grouper iridovirus, GIV)為石斑魚主要的兩種病毒性病原，感染後會導致魚隻大量死亡。NNV在分類上屬於野田病毒科(Nodaviridae)的 β-野田病毒屬(而GIV在分類上屬於虹彩病毒科(Iridoviridae)的蛙病毒屬(Ranavirus)。接種疫苗是預防病毒性疾病的有效策略之一，且雙價疫苗能降低接種疫苗時所耗費的人力與時間成本及免疫期間魚隻受緊迫的次數。本篇研究之目的為開發NNV-GIV雙價疫苗來保護石斑魚抵抗NNV與GIV，並測試NNV-GIV雙價疫苗能否交叉保護魚隻抵抗嘉魶魚虹彩病毒(Red sea bream iridovirus, RSIV)。安全性測試結果顯示，接種NNV-GIV雙價疫苗的魚隻皆活著且食慾正常且免疫魚隻的臟器均未出現組織病變之情況，說明此疫苗對魚隻具有安全性。攻毒試驗結果顯示以NNV及GIV進行攻毒後的免疫魚隻之相對存活率分別為85.1%及86.8%。ELISA結果顯示接種NNV-GIV雙價疫苗能顯著提升魚隻血清中抗NNV、GIV與RSIV之專一性抗體力價。中和試驗結果顯示，接種NNV-GIV雙價疫苗能顯著提升魚隻血清中抗NNV與GIV之中和抗體力價。免疫魚隻的脾臟與頭腎中免疫相關基因的表現量皆有顯著提升，這些免疫相關基因包括TNF-α、IL1-β、MHC-I、MHC-II、CD4、CD8、IgM、IgT、Mx，此結果顯示NNV-GIV雙價疫苗能成功誘發魚隻體內的細胞免疫與體液免疫反應。總結上述研究結果，NNV-GIV雙價疫苗可以有效提升石斑魚的免疫力，保護魚隻對抗病毒的感染。

Grouper is an economically important fish species in aquaculture industry in Asia.Nervous necrosis virus (NNV) and grouper iridovirus (GIV) are two major viral pathogens in groupers and have caused massive mortality. NNV belongs to Betanodavirus genus of Nodaviridae family, and GIV classified into Ranavirus genus of Iridoviridae family. Vaccination is an effective strategy to prevent viral disease, and bivalent vaccines can reduce the cost of both labor and time and decrease the times of stress during vaccination. The purpose of this study was to develop a bivalent vaccine to protect groupers against NNV and GIV, and to investigate whether the NNV-GIV bivalent vaccine could have cross-protection against red sea bream iridovirus (RSIV). The safety test revealed that fish immunized with NNV-GIV bivalent vaccine were all alive and exhibited normal appetite. Moreover, the organs of immunized fish showed no histopathologic effect, indicating that the vaccine is safe for fish. In the challenge test, the relative percent survival (RPS) values of immunized fish were 85.1% and 86.8% in NNV and GIV challenge, respectively. The results of ELISA revealed that immunization with the NNV-GIV bivalent vaccine can significantly increase the titers of specific antibody against NNV, GIV and RSIV in fish sera. The neutralization test revealed that immunization with the NNV-GIV bivalent vaccine can significantly increase the titers of neutralizing antibody in fish sera against NNV and GIV. The expression of immune-related genes in the spleen and head kidney of immunized fish all significantly increased, and these genes includes TNF-α, IL1-β, MHC-I, MHC-II, CD4, CD8, IgM, IgT, and Mx. This result indicated that the NNV-GIV bivalent vaccine could effectively induce cellular and humoral immune responses in fish. In conclusion, the NNV-GIV bivalent vaccine can effectively improve the immunity of grouper and protect the fish against viral infection.