神經壞死症病毒不活化疫苗保護效力評估指標之研究

Abstract

石斑魚是台灣重要的養殖魚種，經濟價值非常高，但病毒性神經壞死症 (viral nervous necrosis disease, VNN) 是造成養殖石斑魚苗及幼魚極高死亡率的重要疾病，VNN的致病原為神經壞死症病毒 (nervous necrosis virus, NNV)。不活化NNV疫苗可以提供石斑魚良好的保護效果，但免疫魚體內的免疫反應的研究資訊仍十分有限。攻毒試驗是測試疫苗保護效力的傳統評估指標，但尋找無病毒帶原的魚及活體實驗皆十分耗時與耗人力，因此本論文擬找出可反應活體攻毒試驗結果的其他保護效力指標。本實驗中，分別將高、低兩種劑量的不活化NNV疫苗，以腹腔注射免疫龍膽石斑幼魚，並在免疫後第4週以肌肉注射方式進行NNV攻毒。高、低劑量免疫魚在攻毒之後的存活率分別為67%及52%，其中高劑量組的存活率顯著高於對照組 (28%)。免疫後第1、2及4週，不活化NNV疫苗誘發的免疫基因表現和對照組之間並沒有顯著性差異，但高、低劑量免疫組的專一性抗體量在免疫後第2及第4週皆顯著性上升。在感染NNV後第3及第5天，高劑量免疫組腦中NNV RNA2量明顯低於低劑量免疫組，且感染後第3天，高劑量免疫組腦中NNV鞘蛋白的免疫組織化學 (IHC) 染色偵測率低於低劑量免疫組。在感染NNV後第3天，免疫組及對照組腦中的IFN-γ、Mx、TNF-α基因表現量顯著上升；CD8α、IgM基因表現量在感染後則是隨著時間逐漸增加。IHC染色的腦組織切片中發現感染NNV後的魚腦有IgM出現，故推測感染NNV會誘發CD8α+淋巴球及IgM+ B淋巴球滲入腦中。另外，死魚腦中IL-1β表現量顯著上升，但此時的病毒量並不是最高峰，推測大量IL-1β表現可能與NNV感染魚的死亡有關。總結所有研究結果，免疫魚的中和抗體力價和NNV攻毒試驗後魚的存活率呈現正相關，因此認為，中和抗體力價可作為評估疫苗保護效力的第二指標。

Grouper aquaculture is economically important in Taiwan, but has suffered from viral nervous necrosis (VNN) disease, which has caused extremely high mortality of fish at larval and juvenile stages. The causative agent of VNN is nervous necrosis virus (NNV). An inactivated NNV vaccine performed good protection efficacy on groupers against NNV infection. However, the information about the immune response of groupers after vaccination with inactivated NNV is still limited. Challenge test is traditionally used for the evaluation of vaccine protection efficacy, but it is time-consuming to find NNV-free groupers and do in vivo test. We therefore attempt to find other suitable indicator(s) for evaluating NNV vaccine protection efficacy. Giant groupers (Epinephelus lanceolatus) were intraperitoneally immunized with high (Vhigh) and low (Vlow) doses of inactivated NNV vaccine, and then intramuscularly challenged with NNV at 4 weeks post vaccination (wpv). The survival rates of Vhigh and Vlow groups were 67% and 52% respectively, and the survival rate of Vhigh group (67%) was significantly higher than that of non-vaccinated group (28%). No immune gene was intensely regulated by the inactivated NNV vaccine at 1, 2 and 4 wpv, but NNV-specific antibodies were effectively induced in both vaccinated groups at 2 and 4 wpv. After NNV infection, the NNV RNA2 load in the brain of Vhigh group was lower than that of Vlow group at 3 and 5 dpi. Moreover, the detection rate of NNV capsid protein in the immunohistochemistry (IHC)-stained brain of Vhigh group was also lower than that of Vlow group at 3 dpi. After NNV challenge, the gene expression levels of IFN-γ, Mx and TNF-α in brains of vaccinated and control groups were up-regulated at 3 dpi; CD8α and IgM gene expressions gradually increased during infection period. In addition, IgM signals appeared in the NNV-infected brains after IHC staining, suggesting that CD8α+ lymphocytes and IgM+ B lymphocytes may infiltrate into the brain after NNV infection. IL-1β gene expression was significantly high in the brain of dead fish at 8-12 dpi; however, the viral load in the brain did not peak at the same time, suggesting that the death of NNV-infected groupers may due to the over-expression of IL-1β. In conclusion, neutralizing antibody titers in the serum of vaccinated and control groups were associated with their survival rates after NNV infection, it is therefore considered to be a suitable evaluation indicator for the protection efficacy of NNV vaccine.