點帶石斑魚經疫苗接種後其對抗虹彩病毒感染 之基因表現與免疫反應

Abstract

石斑魚虹彩病毒─可感染石斑魚生活史各個階段，魚隻罹病後容易死亡，常導致石斑魚育成率降低，並造成經濟上的損失。對於虹彩病毒的防範，開發一個有保護力的疫苗，並對疫苗效力的研究與評估是各方所關注的重點。過去，在評估疫苗的效力，通常僅能鎖定專一性抗體 (specific antibody) 的效價高低與持續的時間，並搭配活毒攻毒後的相對存活率 (relative percent survival, RPS) 做為評估依據。但對於病毒與宿主間交互作用的基礎研究仍然缺乏。本實驗室先前已藉由研究微陣列晶片 (microarray) 的結果，篩選出虹彩病毒感染石斑魚後，38個具有兩倍以上正調控，48個兩倍以上負調控的基因群，並進一步以RT-qPCR確認感染虹彩病毒後不同時間點，魚隻的頭腎、脾臟基因表現變化，共分析出30個虹彩病毒反應基因 (virus responsive genes, VRGs) ，參與宿主先天性免疫和後天性免疫反應。 本研究以福馬林不活化 GIV 疫苗，對於點帶石斑魚苗進行腹腔免疫注射後，再進行虹彩病毒活毒攻毒測試。分析相關的15個 VRGs 在病毒攻毒後的表現變化量。其中 DHX58、HECT、Mx、Viperin、ISG15、PLAUR、CD9、VLIG1 等基因表現量在感染過後第一天到第三天的頭腎及脾臟中有上升趨勢，在第七天之後逐漸下降，這些免疫基因的調控與先天性免疫系統中的干擾素傳遞路徑非常符合。此外在本研究所分析的基因之中，攻毒過後大多數的基因表現量在疫苗注射組是低於控制組的，這樣的現象與魚體內 GIV MCP (major capsid protein) 的表現量有正比的關係，顯示經疫苗免疫後的魚隻確實產生專一性抗體，可以中和 (neutralize) 病毒減低病毒量。攻毒後 30 天的觀察結果，在免疫注射組的存活率 100 %，相較於控制組的 0 %，達到 100 % 的相對存活率 (relative percent survival, RPS) ，證實所施打的疫苗確實具有保護效力。 在施打兩種不同劑量的疫苗過後，魚血清內專一性抗體 (specific antibody) 的追蹤實驗，也發現無論在高或低劑量免疫注射組別之中，抗體於一周後就攀升到最高，持續到第八周有些微下降之趨勢，而專一性抗體生成的量對於注射進去魚體內的疫苗有劑量正比 (dose-dependent) 關係。

Grouper iridovirus (GIV) can infect all life stages in grouper and cause serious death. It also make low survival rate in grouper from fry to juvenile stage, and may cause aquaculture economic damage. To develop a high efficacy vaccine is important to resist iridovirus . Calculating the relative percent survival (RPS) rate post challenge and detecting the titer of specific antibody and its maintain efficiency, are used to estimate the efficacy of vaccine in the past. But the interaction between host and iridovirus is not clear completely. According microarray results in GIV infection orange-spotted grouper (Epinephelus coioides) , we selected many candidate gene in our lab previously. Thus, we have confirmed and analyzed 30 virus responsive genes (VRGs) in response to innate immunity and adaptive immunity in grouper’s head kidney and spleen after GIV infection. In this study, we analyzed the expression of 15 VRGs in grouper following vaccination against GIV infection by intraperitoneal injection. We discovered that DHX58, HECT, Mx, Viperin, ISG15, PLAUR, CD9 and VLIG1 expression patterns are very similar, and these genes are considered to involving in interferon regulated pathway in innate immunity. On the other hand, most VRGs expression are lower in vaccination group than in control after GIV challenge. The maximum anti-GIV specific antibody is produced in fish sera post vaccination one week, even in the group of low dose vaccine treatment. The ELISA results showed that host immune response by vaccination exist dose-dependent relationship. These results indicated that the neutralization between ant-GIV specific antibody and GIV caused viral load reducing in the vaccinated group after GIV challenging.