池塘养殖斑鳢弹状病毒的分离与初步鉴定

ISOLATION AND PRELIMINARY IDENTIFICATION OF RHABDOVIRUS FROM CHANNA MACULATA CULTURED IN POND

从患暴发性死亡的斑鳢病灶中分离出一株弹状病毒。取病鱼肝、脾、肾组织过滤液接种EPC、FHM细胞,连续传至第3代后28℃培养35h出现细胞病变(CPE),测得病毒半数细胞感染量为10 5.746/0.1 mL。将病变细胞制成超薄切片,透射电镜下观察到细胞质中存在大量呈子弹状的病毒颗粒,大小约53 nm×140 nm。用上述组织过滤液及F3代细胞培养病毒液回归感染健康斑鳢均能显示与自然发病鱼相似的症状,死亡率达100%。根据鳜鱼弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)N蛋白保守序列设计的引物对斑鳢病毒的基因组RNA进行RT-PCR扩增,得到大小约400 bp的阳性片段。对该片段克隆、测序后与GenBank中序列进行BLAST比对,发现该基因序列与SCRV同源性最高,为94%。选取GenBank中已登录的病毒性出血败血症病毒(VHSV)、鳢弹状病毒(SHRV)、鲤春病毒血症病毒(SVCV)、鳜鱼弹状病毒(SCRV)、传染性造血器官坏死病毒(IHNV)、狂犬病毒(RV)、牙鲆弹状病毒(HIRRV)相关序列构建系统进化树,结果表明,该基因序列与SCRV聚为一支。由于该病毒粒子的形态大小与SCRV(100 nm×200 nm)存在一定差异,暂将其命名为斑鳢弹状病毒(CHRV)。

During September,2010,an unknown disease with highly mortality of over 80% broke out in cultured Channa maculata,at Foshan area of Guangdong province.A strain of rhabdovirus was isolated from the diseased Channa maculata.After the third passage,obvious cytopathic effect（CPE） could be observed in EPC and FHM,inoculated with the ultra-filtrate of liver,spleen and kidney from diseased fishes with no bacteria.Mass of bullet-shaped viruses which about 53 nm×140 nm in size,were observed in the ultrathin sections made of infected EPC under electron microscopy.Both artificial infection for healthy Channa maculata with either filtrate of tissues or infected EPC by intraperitoneal injection caused 100% mortality.The symptoms of the infected fishes were similar to those of natural infection.This result indicated that this virus was the lethal pathogen of the fulminant disease.With the primers designed according to the conserved regions of SCRV N gene,specific products with predicted size（400 bp） were obtained from all the diseased tissues and infected cells.Comparative analysis of nucleotide sequences was performed with the GenBank databases using BLAST database network service.The results showed that the gene products shared a highest identity（i.e.,94%） with the corresponding sequence of SCRV [EF575725.1].On the basis of neighbor-joining analyses of the N gene sequences,phylogenetic tree was constructed with the Clustalx and Mega 4,and it formed a single cluster with SCRV.As their sizes and symptoms of the diseased fishes had some differences,the virus in present research was named as CHRV tentatively.