魚類野田病毒與巨大細胞病毒雙價疫苗對龍膽石斑魚保護效果之評估

Abstract

龍膽石斑魚（Epinephelus lanceolatus）為亞洲高經濟價值的養殖魚種，常遭受魚類野田病毒與巨大細胞病毒的感染，而造成養殖業重大的損失，本研究開發的不活化雙價疫苗選用赤點石斑魚神經壞死症病毒（Red-spotted grouper nervous necrosis virus, RGNNV）以及嘉鱲魚虹彩病毒（Red seabream iridovirus, RSIV）製成，其分別屬於β野田病毒屬及巨大細胞病毒屬，並於龍膽石斑魚上測試此疫苗的保護效果，龍膽石斑魚腹腔接種此雙價疫苗，第二週施打同劑量之第二劑，第六週進行兩種病毒的攻毒試驗。在免疫後四週，石斑魚器官的組織切片在免疫組與對照組沒有差異，說明疫苗具備安全性。攻毒試驗結果顯示，免疫此雙價疫苗的魚隻在NNV與RSIV攻毒後的相對存活率為87%及100%，且免疫後可誘發抗NNV及RSIV的特異性抗體及中和抗體。在接種疫苗後零、一、二、三、六週採集魚隻頭腎及脾臟檢測免疫基因之表現，結果顯示頭腎及脾臟的IgM、IgT、TNF-α、IL-1β及Mx基因表現在免疫後皆有上升，表示疫苗免疫後能刺激石斑魚的免疫反應。此外，亦測試NNV-RSIV雙價疫苗免疫魚隻後，是否具有對抗石斑魚虹彩病毒（grouper iridovirus, GIV）的交叉保護效果，結果顯示，免疫魚攻毒後的相對活存率皆低於40%，雖然免疫魚隻血清中有測得GIV特異性抗體，但無法檢測出對GIV的中和抗體，因此， NNV-RSIV疫苗對抗GIV的效果較低。總結，不活化NNV-RSIV雙價疫苗接種於龍膽石斑魚是安全的，且能有效保護魚隻抵抗NNV及RSIV的感染。

The giant grouper（Epinephelus lanceolatus）is a cultured fish species with high economic value in Asia, which is often infected by fish nodavirus and megalocytivirus, causing mass losses in the aquaculture industry. In this study, an inactivated bivalent vaccine was developed using red-spotted grouper nervous necrosis virus (RGNNV) and red seabream iridovirus (RSIV) which respectively belong to the genera of betanodavirus and megalocytivirus. The protective effect of this vaccine was tested on the giant groupers. The giant groupers were intraperitoneally immunized with this bivalent vaccine and then inoculated the 2nd injection with the same dose at 2 weeks post vaccination (wpv). Challenge with these two viruses was performed at 6 wpv. The tissue sections of grouper organs at 4 wpv showed no difference between immunized and control groups, which indicated that the vaccine was safe for groupers. The results of challenge tests showed that the relative percent survival (RPS) rates of the bivalent vaccine against NNV and RSIV were 87% and 100%. Moreover, specific and neutralizing antibodies against NNV and RSIV were induced after immunization. The head kidney and spleen of the fish were collected at 0, 1, 2, 3, and 6 wpv to analyze the immune gene expression. The results revealed that the gene expression of IgM, IgT, TNF-α, IL-1β, and Mx in the head kidney and spleen increased after immunization, which indicated that the vaccine stimulated immune responses in groupers. Additionally, the cross-protective effect of NNV-RSIV bivalent vaccine against grouper iridovirus (GIV) was also test. The result showed that the RPS was < 40%. Although the specific antibodies against GIV were detected in the serum of immunized fish, no anti-GIV neutralizing antibodies was detected. Therefore, the protective effect of NNV-RSIV vaccine against GIV was lower. In conclusion, the inactivated NNV-RSIV bivalent vaccine is safe to immunize in the giant groupers and can efficiently protect fish against NNV and RSIV infections.