病毒性神經壞死症(Viral nervous necrosis disease,VNN disease)為近年來臺灣石斑養殖上極為嚴重的疾病，其致病原為神經壞死病毒(Nervous Necrosis Virus)，此病毒感染易造成石斑魚苗及幼魚大量死亡，死亡率往往高達100％，造成臺灣石斑養殖業極大的損失。該病毒帶有兩條單股RNA，分別為RNA1與RNA2，其中RNA1的功能為RNA-dependent RNA polymerase；RNA2則可轉譯出病毒的外殼蛋白質。本研究室於2002年至2004年期間，至台南及高雄地區數個石斑魚養殖場中收集罹患病毒性神經壞死症之病魚，其中包含各種臺灣地區熱門養殖石斑魚種:點帶石斑、龍膽石斑以及金錢斑；另一方面，利用SYBR Green I chemical，我們發展檢測NNV之螢光即時PCR(real-time PCR)系統，其偵測極限可達到190病毒copies，和傳統RT-PCR相比，其靈敏度高於十倍。藉由此系統進行NNV在罹病龍膽石斑魚組織分佈的定量，發現NNV存在於各器官中，但含量有顯著差異，其中以腦部及眼睛最多。台灣石斑魚養殖產業在病毒與細菌等病原肆虐下，若能有良好的檢測系統協助，瞭解其致病機制與傳染的途徑，就是一件非常重要的工作。因此本實驗室擬藉由已開發完成對於神經壞死病毒高靈敏度及早期發現的快速即時檢測方法，來瞭解神經壞死病毒致病機制與傳染途徑傳染的相關途徑或宿主。 In recent years, viral nervous necrosis disease （VNN）was the greatest disease of hatchery-reared grouper in Taiwan . The causative agent of VNN was nervous necrosis virus （NNV）and the mortality of grouper larvae and juvenile during disease outbreak were usually above 90％. NNV contains two, single stranded, positive-sense RNA called RNA1 and RNA2. RNA1 encodes RNA dependent RNA polymerase, RNA2 encodes viral coat protein. In our team, we collected VNN diseased grouper samples from several aquaculture farms near Tainan from 2002 to 2004 which including orangespotted grouper（Epinephelus coioides）, giant grouper（Epinephelus lanceolatus） and potato grouper（Epinephelus tukula）. Therefore, we also developed a real-time quantitative PCR diagnosis system for NNV, the detectable limit was 190 virus copies, and the sensitivity was 10 times than conventional PCR detection. By using real-time quantitative PCR detection, we knew that NNV could be found in many organs, but the virus quantity in brain and eyes were usually 1000 times than other organs in Giant grouper. Until now, the grouper farming industry has a big problem of pathogens infection, especially the larva stage of grouper. If we has a good diagnosis method to detect the virus or bacteria, we can early find and prevent the disease through the pathogenic mechanism and transmission pathway. In this project, we will use the developed real-time PCR diagnosis system that has sensitivity and specificity for NNV detection to determine the pathogenic mechanism and transmission pathway of NNV.
