台灣沿海野生魚種帶原結病毒的田野調查與分析

Abstract

屬於魚類結病毒(Fish nodaviruses)的神經壞死症病毒(Nervous Necrsis Virus, NNV) 會造成養殖石斑幼苗大量死亡，而魚類虹彩病毒，包括台灣石斑虹彩病毒（Grouper Iridovirus of Taiwan, TGIV）及嘉鱲魚虹彩病毒（Red Sea Bream Iridovirus, RSIV），則是造成石斑幼魚及成魚陸續死亡的重要病原體。本實驗目的在建立神經壞死症病毒及魚類虹彩病毒的快速核酸檢測法，並分析在不同季節與海域所捕撈的野生魚種及養殖魚種中帶原NNV的情況。本研究成功地開發了適用NNV的迴路媒介恆溫增殖法（Loop-mediated isothermal amplification assay, LAMP）的核酸引子與反應條件，並建立同時檢測兩種魚類虹彩病毒的Multiplex PCR所需要的各核酸引子及藥品濃度，為將來田野調查提供了更簡便、快速又精準的檢測工具。在台灣沿海所採集的75種魚當中，其中30種魚有NNV帶原的情形。在南部海域，於各季節所採集的魚體中都有帶原NNV的情況。南部養殖的石斑苗，自春末至夏末常有NNV造成魚苗大量死亡的疫情；然而，烏魚卻在秋冬兩季才有NNV的高檢出率。

Nervous necrosis viruses (NNV), belonging to fish nodavirus, have caused mass mortality of grouper larvae. Fish iridoviruses, including grouper iridovirus of Taiwan (TGIV) and red sea bream iridovirus (RSIV), are important pathogens for the continuous death of grouper juveniles and adult fish. One aim of the present study is to develop rapid and sensitive nucleic acid-based diagnosis methods for fish nodavirus and two kinds of fish iridoviruses (TGIV, RSIV). Another aim of this study was to screen NNV in wild and cultured fish collected from different aquatic areas and seasons. We successfully established six NNV-specific primers and reaction program for loop-mediated isothermal amplification assay (LAMP), and the proper concentration of primers and chemicals for simultaneous amplification of nucleic acids of TGIV and RSIV in Multiplex PCR. By RT-PCR and nested-PCR screening of NNV, 30 of 75 species of wild fish collected from four season and different coasts of Taiwan were NNV carrier, especially the wild fish from southern area. Mass mortality of cultured grouper larvae by NNV infection occurred since late spring to the end of summer. However, NNV was only detected in the mullets collected during autumn and winter.