6株鱼类病毒性神经坏死病病毒cp基因的分子特征和遗传发生分析

Molecular Charaterization and Phylogenetic Tree of cp Gene from Six Isolates of Viral Nervous Necrosis Virus

根据已报道的鱼类病毒性神经坏死病毒（viral nervous necrosis virus,VNNV）衣壳蛋白基因（coatprotein gene,cp基因）设计引物,对2006年从我国沿海养殖石斑鱼中分离到的6株鱼类神经坏死病毒进行RT-PCR扩增,特异性扩增出6株病毒的完整衣壳蛋白基因,分别连接到pMD18-T载体中,并进行序列测定和分析。结果发现,6个VNNV分离株的cp基因核苷酸序列的同源性在97.3%以上;推导的氨基酸序列同源性在79.6%～99.1%之间。根据cp基因核苷酸序列绘制分子进化树,发现6个VNNV分离株在进化树同一分枝上,属于RGNNV基因型,与已报道的点带石斑鱼神经坏死病病毒（ECNNV）、巨石斑鱼神经坏死病病毒（ET-NNV）及玛拉巴石斑鱼神经坏死病病毒（MGNNV）亲缘关系较近。试验结果表明,目前我国沿海养殖场发生的鱼类病毒性神经坏死病均由同一来源的病毒引起,该基因型的鱼类神经坏死病毒在我国沿海养殖的石斑鱼中广泛传播流行。

Six viral nervous necrosis virus （VNNV） were isolated from cultured yellow grouper in 2006 in the shore of China. Total viral RNA genome was extracted. Based on the complete sequences of RNA2 which have been submitted to GenBank, a pair of primers were designed and the coat protein genes from the 6 isolates were amplified by means of reverse-transcriptase polymerase chain reaction （RT-PCR）. The RT-PCR products were ligated into the pMD18-T vector and transformed into E. coli DH5a. The recombinant plasmid were sequenced respectively. There was more than 96. 1% nucleotide identityies and 79. 6%-99. 1% among the deduced amino acid sequences within the six isolates. Phylogentic analysis showed that the isolates shared evolutionary direction with EVNNV, ETNNV and MNNV. It was revealed that these isolates bdonged to genotype RGNNV. It was deduced that the strains of RGNNV genotype were dominant and prevalent in spotted grouper cultured in China during the past several years.