Histopathological Study of Viral Nervous Necrosis and Characterization of Nervous Necrosis Virus in Hatchery-Reared Grouper

• Main Authors: Hsien-Chieh Chiu, 邱顯傑
• Other Authors: Ing-Cherng Guo
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/73649926674369422545

碩士 === 國立臺灣大學 === 獸醫學研究所 === 88 === In recent years, viral nervous necrosis (VNN) caused mass mortalities of hatchery-reared grouper larvae in southern Taiwan. The disease was characterized by vacuolation in the spinal cord, brain and retinal tissue in histopathological examination. By using two pairs of specific primers (F1-R3 & F2-R3), the RT-PCR had amplified two predicted DNA fragments (T2 870 bp & T4 421 bp) from RNA extracts of diseased grouper larvae. The homology (78 %-98 %) of T2 fragment to that of the other nervous necrosis viruses (NNV) such as SJNNV, TPNNV, BFNNV, JFNNV and RGNNV demonstrated that the diseased fish were infected with a novel strain of NNV, named YGNNV (yellow grouper nervous necrosis virus). Cytopathic effects (CPE) were observed in a grouper fin cell line, which was infected with extracts from diseased grouper larvae. The pure virus particles were harvested from CPE cells by CsCl gradient centrifugation. By examination of transmission electron microscopy, the round-shaped and non-enveloped virus particles with diameters of 25-27 nm were observed. The virions in CPE cells were found intracytoplasmically in paracrystalline arrays and membrane-associated aggregates. The SDS-PAGE resolved two major proteins (100 kDa, 43 kDa) of pure virus particles. The T2 and T4 DNA fragments encoding partial coat protein of NNV had been cloned into prokaryotic expression vector and overexpressed in E. coli BL21(DE3) pLysS strain. Two recombinant coat preteins, 35 kDa and 16 kDa, were purified by Ni2+-NTA affinity column. Anti-NNV monoclonal antibody was used to define the tissue tropism of NNV by immunohistochemistry. Positive signals were restricted in the neural tissue such as spinal cord, brain and retina, in good agreement with the histopathological examination. This study demonstrates the typical symptoms of VNN and the characters of NNV in grouper larvae from southern Taiwan.