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2013年4月,广东省一锦鲤养殖场暴发不明病因疾病,濒死锦鲤在塘边游动缓慢直至死亡,死亡率高达100%。现场采样发现,发病锦鲤体长25 cm,眼球凹陷,胸鳍及腹鳍出现出血斑点,解剖发现内脏器官包括肝、脾、肾肿大。细菌分离结果显示,内脏器官肝脏和肾脏中未分离到细菌。提取自然发病鱼的肝、脾、肾、鳃组织DNA作为模板,采用世界动物卫生组织(OIE)推荐的锦鲤疱疹病毒(KHV)检测引物进行PCR扩增,均能扩增出预期大小的特异性产物。NCBI的Blast搜索结果显示,扩增序列与KHV胸苷激酶(thymidine kinase,TK)基因核苷酸序列同源性为99%。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾锦鲤,可复制出与自然发病相似的症状,并于7 d内全部死亡。取病鱼的鳃和肾脏研磨过滤除菌后进行细胞感染实验,结果显示,组织滤液感染CCB细胞后,盲传5代可以观察到典型的细胞病变效应(CPE)。将出现典型CPE的CCB细胞进行超薄切片制备和电镜观察,电镜下病毒呈对称20面体,直径约100 nm。将出现典型CPE的细胞进行间接免疫荧光实验,可以观察到特异性荧光。根据TK基因全长序列建立系统进化树,证实该毒株为KHV亚洲型毒株,暂命名为KHV-GZ1301株。研究结果可为KHV起源进化、分类以及疾病防控提供重要材料。
In April 2013, an unidentified etiological disease was reported in a koi carp farm in Guangdong Province. The koi carp swimming slowly and died at the pond with a mortality rate of 100%. Spot sampling found that the incidence of Koi body length 25 cm, eyeball depression, pectoral and pelvic fins bleeding spots found anatomical organs, including liver, spleen, enlarged kidney. The results of bacterial isolation showed that no bacteria were isolated from the liver and kidney of internal organs. DNA from liver, spleen, kidney and gill of naturally occurring fish was extracted and used as template to amplify the expected size by PCR using primers of KHV recommended by the World Animal Health Organization (OIE) Sexual product. The NCBI Blast search results showed that the nucleotide sequence homology between the amplified sequence and the KHV thymidine kinase (TK) gene was 99%. After diseased fish internal organs were ground, filtered and sterilized, 20 koi carps injected intraperitoneally were able to replicate the symptoms similar to the natural ones and all died within 7 days. Gills and kidneys from diseased fish were grinded and sterilized. Cell infection experiments were performed. The results showed that the typical cytopathic effect (CPE) was observed after blind passage of CCB cells for 5 generations. CCB cells with typical CPE were prepared by ultrathin section and electron microscopy. The electron microscopy showed that the virus was symmetric 20-sided with a diameter of about 100 nm. Indirect immunofluorescence experiments were performed on cells with typical CPE, and specific fluorescence was observed. Phylogenetic tree based on the full-length TK gene sequence confirmed that the strain was KHV Asian strain, tentatively named KHV-GZ1301 strain. The results provide important materials for the evolution, classification and disease control of KHV.