| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 93 === Abstract
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 study, we collected VNN diseased grouper samples from several aquaculture farms near Tainan from 2002 to 2004 which including orange-spotted grouper（Epinephelus coioides）, giant grouper（Epinephelus lanceolatus） and potato grouper（Epinephelus tukula）.We cloned RNA1 sequences from 9 isolates and RNA2 sequences from 13 isolates. Then using Clustal W program to compare the amino sequences. The result showed that according to the deduced amino acid of RNA1 gene, there were 8 variation strains out of 9 isolates and with an amino acid identity of 97.2％ to 99.9％ between these strains. On the other hand, according to the amino acid sequences of NNV capsid protein, there were 2 variation strains out of 13 isolates. The two strains with an amino acid identity of 99.6％ to 100％, and share the same point mutation site （a.a. 214）. All the 13 isolates belong to RGNNV genotype.
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. The virus propagation test in vitro showed that grouper fin cell line（GF-1）could propagate 100 to 1000 times virus particles than liver and kidney cell lines, these two results indicate that there were tissue tropism when virus infected cell and propagated. Finally, we chose 5 RNA1 variation strains to test their replication ability, but there was no significant difference between these strains.
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