斜带石斑鱼(Epinephelus coioides)病毒性神经坏死病毒的纯化分析及检测方法建立

The purification and detection methods research of viral nervous necrosis in orange-spotted grouper Epinephelus coioides

根据GenBank中已有的鱼类病毒性神经坏死病毒(NNV)RNA2基因序列,设计引物,从福建厦门具有典型NNV发病症状的斜带石斑鱼中克隆了RNA2基因的全长序列,并将序列提交到GenBank获得登录号为MF510920,命名为XMNNV。系统进化树分析结果表明XMNNV与RGNNV聚类在一起,与SJNNV、BFNNV和TPNNV等其他鱼类神经坏死病毒亲缘关系较远,说明本研究分离得到的XMNNV属于RGNNV基因型。通过超速离心方法对病毒进行提纯,得到了纯化的NNV病毒。电镜观察结果表明,病毒粒子直径20～25 nm,结构为正二十面体,与已经报道的NNV结构一致。通过对XMNNV与其它RGNNV RNA2基因进行序列比对,在保守区设计引物,运用RT-PCR方法建立了RGNNV的PCR检测方法,该方法灵敏度高达67 copies/μL。纯化的病毒对E11(条纹月鳢细胞系)和大黄鱼肌肉细胞进行感染,结果表明该病毒可以感染这两种鱼类细胞。E11细胞被感染病毒后,细胞出现空泡化,并最终导致细胞分解死亡;大黄鱼肌肉细胞感染后,细胞变圆,慢慢从培养皿壁脱落,最终解体死亡。另外,对感染后细胞进行PCR检测,结果显示为阳性,进一步确定了分离的NNV具有感染这两种细胞的能力。本研究通过电镜观察和PCR检测两种方法确定了患病石斑鱼携带NNV,通过对两种鱼类细胞的感染实验,确定了该病毒具有一定的感染能力。综上,本研究为石斑鱼NNV疾病的诊断提供了有效的方法,对石斑鱼NNV疾病的预防具有一定的指导意义。

Primers were designed based on various grouper nervous necrosis virus RNA2 genome sequence obtained from GeneBank.The coat protein gene of Fujian Xiamen Epinephelus coioides nervous necrosis virus was cloned and sequenced,while the sequence was submitted to the GenBank database under accession number MF510920.In the phylogenetic tree,the coat protein gene from XMNNV formed distinct group with that from RGNNV,but separated to the SJNNV,BFNNV and TPNNV nervous necrosis virus.Based on the result of molecular phylogenetic analysis,XMNNV was found belonged to red-spotted grouper nervous necrosis virus(RGNNV)genotype.The virus particles were purified by ultracentrifugation.The results showed that it was an icosahedral virus with a mean diameter of 20~25 nm by electron microscopy,consistent with the reported structure of NNV.According to the sequence alignment results of XMNNV and the other RGNNV RNA2,the specific primers were designed according to the highly conservative sequence of capsid protein gene of viral nervous necrosis virus.A reverse-transcription polymerase chain reaction(RT-PCR)assay for fish nervous necrosis virus was developed,the sensitivity of the method was 67 copies/μL.The isolated virus was used to infect E11 cell line and Larimichthys crocea muscle cells,and both cells showed sensitive for the isolate.While incubated with E11 cell,CPE was developed and vacuolation after incubation. L.crocea muscle cell was round and shedding off the wall,when broken up and died eventually.Besides,the infected samples were cloned and sequenced.The result of PCR indicated that the isolated virus was NNV.According to the transmission electron microscopy and PCR assay,we suspected the E.coioides was infected by red-spotted grouper nervous necrosis virus.This research provided effective method for the diagnosis and will be used in the field of prevention of grouper disease.