遭受神經壞死病毒及石斑魚虹彩病毒感染而影響之點帶石斑基因表現分析

Transcriptional analysis of orange-spotted grouper (Epinephelus coioides) either in nervous necrosis virus or grouper iridovirus infection

吳明山(Ming-Shan Wu)

指導教授 ： 曾晴賢 ； 張繼堯

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2012年)

摘要

石斑魚為台灣地區重要的養殖魚種，近年來飽受神經壞死病毒（nervous necrosis virus，NNV）以及虹彩病毒（iridovirus）侵害，造成大量魚苗及成魚死亡而影響養殖漁業甚鉅。過去二十多年，全世界有許多硬骨魚種遭受一種名為神經壞死病毒的魚類野田病毒危害甚深，該病毒會造成仔魚及稚魚嚴重的病毒性神經壞死、病毒性腦病變以及病毒性視網膜病變。雖然遭 NNV 感染的魚苗死亡率幾近 100%，仍有少數魚隻在此浩劫殘存。為了瞭解這些殘存者在分子生物學上的差異性，我們以在傳統養殖場中歷經 NNV 爆發而倖存的點帶石斑仔魚做為實驗對象，研究其基因上的差異性。藉由一個具備 9600 clones 的點帶石斑仔魚 cDNA 微陣列晶片，比較倖存的仔魚（依所測得的 NNV 含量區分三群）和受感染的仔魚之間基因的差異性，並進一步以定量即時聚合酶鏈鎖反應結果驗證。相較於染病仔魚，在倖存仔魚中表現量較高的基因包括：adenylate kinase 1-2，myosin binding protein H-like，myosin light chain 2，myosin light chain 3，tropomyosin，fast/white muscle troponin T embryonic isoform 以及 parvalbumin 1、2 型；而 apolipoprotein A-I，trypsinogen，pyruvate kinase以及 astacin-like metalloprotease 則在染病仔魚中的表現量較高。此外，雖然多數免疫因子在染病仔魚中的表現量較高，免疫球蛋白 M（immunoglobulin M，IgM）重鏈基因在倖存但有高病毒含量的仔魚中卻有著比染病仔魚更高的表現量，暗示著體液免疫也提供宿主保護而免遭病毒感染的危害。這些結果指出在免疫系統尚未發育完全的仔魚變態階段，部分非免疫相關的基因可能在遭受神經壞死病毒感染後仍可倖存的事件中扮演重要角色。
在另一方面，石斑魚虹彩病毒（grouper iridovirus，GIV）亦會造成大規模石斑魚死亡而導致嚴重的經濟損失。藉由點帶石斑腎臟 cDNA 微陣列晶片，我們篩選出 38 個在 GIV 感染點帶石斑後表現增加的基因，以及 48 個表現降低的基因。我們挑選其中 24 個基因，並額外加入 7 個免疫相關因子，在頭腎及脾臟中做進一步的定量表現確認。有 30 個基因於 GIV 感染後在脾臟中表現會有顯著性增加；而在頭腎中只有 23 個表現增加的基因，另外有 5 個基因的表現則是降低。我們同時建立這些基因在脂多醣體（LPS）和人工合成雙股核醣核酸類似物（PIC）刺激下的轉錄表現特徵。比較後發現，多數基因在 PIC 處理後的轉錄表現特徵與 GIV 刺激後較為相似。其中有 7 個基因由於表現模式相近而共同被歸類為干擾素相關因子：RNA helicase DHX58，ISG15，viperin，HECT E3 ligase，CD9，urokinase plasminogen activator surface receptor（PLAUR）以及 Mx-1。以不活化 GIV 進行免疫動作，在頭腎與脾臟中可見 DHX58，viperin，IL-1beta，IL-8，COX-2，HECT E3 ligase，PLAUR，IgM，Mx-1，very large inducible GTPase-1及 TNF-alpha表現有被顯著誘發，末列6個基因在追加免疫後更有逐漸增加表現的情形。這些基因或許在適應性抗病毒反應中扮演了重要角色。總結來說，我們獲得了數個病毒反應基因並建立其時間性反應型態，同時確認數個潛在免疫標誌基因以提供未來在魚類抗病毒防禦機制上更深入的研究。

關鍵字

石斑 ； 點帶石斑 ； 神經壞死病毒 ； 虹彩病毒

並列摘要

Groupers (Epinephelus spp.) are important aquaculture fish species in Taiwan, but they are highly susceptible to nervous necrosis virus (NNV) and iridovirus which often cause significant economic losses to grouper aquaculture. NNV, a piscine nodavirus, has caused serious viral nervous necrosis and viral encephalopathy and retinopathy in hatchery-reared larvae and juveniles of a wide range of teleost species worldwide in the last two decades. Although the mortality of NNV-infected larvae is nearly 100%, there are still some larvae that survive this catastrophe. To comprehensively understand the variations of these survivors at the molecular level, we collected orange-spotted grouper (Epinephelus coioides) larvae that survived an NNV outbreak in an indoor hatchery in southern Taiwan to study differential gene expression. Survived larvae with high, medium and low levels of detected NNV were compared with morbid larvae using a 9600-clone-containing grouper larva cDNA microarray, and differential gene expression was further confirmed by a quantitative real-time polymerase chain reaction. Significant gene expression variation exists in survived larvae. The following genes were confirmed that had relative 2-folde higher expression in survived larvae: adenylate kinase 1-2, myosin binding protein H-like, myosin light chain 2, myosin light chain 3, tropomyosin, fast/white muscle troponin T embryonic isoform, and two types of parvalbumin genes. Four genes were confirmed that expressed higher relatively in morbid larvae: apolipoprotein A-I, trypsinogen, pyruvate kinase, and astacin-like metalloprotease. Moreover, although most immune factors had higher expression in morbid larvae, immunoglobulin M (IgM) heavy chain gene transcription was significantly higher in survived larvae that carried high virus levels than morbid larvae, indicating that humoral immunity might protect organisms from viral infection. These results suggest that some non-immune-related genes may have played important roles in the survival event during the immune-immature larval metamorphosis stage after NNV infection.
In the other hand, disease caused by grouper iridovirus (GIV) also has resulted in economic losses due to high mortality in all stages of grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1beta, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-alpha in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.

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