鱼病毒性神经坏死病病毒(VNNV)不同基因型鉴别方法的建立及在VNN检疫和监测中的应用

Establishment of discriminating method of various genotypes of viral nervous necrosis virus (VNNV) and application in quarantine and surveillance of VNNV

从GenBank中查找出乙型野田村病毒组中海水鱼类各病毒的序列,并用Sequencher多重序列比较软件将其分到条纹神经坏死病毒(stripedjacknervousnecrosisvirus,SJNNV)组、条纹星鲽神经坏死病毒(barfinfloundernervousnecrosisvirus,BFNNV)组、红点石斑鱼神经坏死病毒(redspottedgroupernervousnecrosisvirus,RGNNV)组和虎斑东方神经坏死病毒(tigerpuffernervousnecrosisvirus,TPNNV)组4个基因型的组别中。用DNAsis序列比较软件比较同一基因型各基因序列之间的同源性,均在85%以上;不同基因型之间序列的同源性,均在66%以下。结合Premier引物设计软件和Sequencher序列多重比较软件,设计了4对引物,采用逆转录聚合酶链式反应(RT PCR)来鉴别这4个不同的基因型。对从深圳口岸进境的产地为台湾的海水鱼苗和广东、福建两省养殖的主要海水鱼类进行检疫和监测,结果在进境的海水鱼苗中检出有RGNNV基因型的VNNV,在福建和广东省养殖的石斑鱼成鱼和鱼苗的病鱼体内均检测到RGNNV基因型的VNNV,对上述扩增产物基因序列进行比较,相似性均在96.5%以上,推导出的氨基酸序列与玛拉巴石斑鱼神经坏死病毒(MNNV)序列相似性均为100%。

All of the sequences of β-nodavirus were collected from GenBank and divided into four genotype groups, SJNNV (Striped jack nervous necrosis virus) group, BFNNV (Barfin flounder nervous necrosis virus, BFNNV) group, RGNNV (Redspotted grouper nervous necrosis virus) group and TPNNV (Tiger puffer nervous necorsis virus) group. The similarity among the sequences in the same genotypes was more than 85% and the similarity among different genotypes was less than 66%. Four pairs of primers were designed using the softwares of Premier and Sequencher. The four genotypes were identified and discriminated by RT-PCR with these primers. Quarantine and supervision were conducted on the larvae of marine fish imported from Taiwan through Shenzhen port and the marine fish cultured in Guangdong and Fujian provinces. The VNNV of RGNNV genotype was detected in the samples of imported larvae of marine fish. The same genotype was also detected in diseased adult fish and larvae of groupers (Epinephellus sp.) cultured in Guangdong and Fujian provinces. The similarity of gene sequences among all of these amplification products was more than 96.5% and the similarity of deduced amino acid sequences between these products and Malabaricus nervous necrosis virus (MNNV) was 100%.