CAJ | 학술논문

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)。
종환폭발성사망적반례병조중분리출일주탄상병독。취병어간、비、신조직과려액접충EPC、FHM세포,련속전지제3대후28℃배양35h출현세포병변(CPE),측득병독반수세포감염량위10?5.746/0.1 mL。장병변세포제성초박절편,투사전경하관찰도세포질중존재대량정자탄상적병독과립,대소약53 nm×140 nm。용상술조직과려액급 F3대세포배양병독액회귀감염건강반례균능현시여자연발병어상사적증상,사망솔체100%。근거궐어탄상병독(Siniperca chuatsi rhabdovirus, SCRV)N단백보수서렬설계적인물대반례병독적기인조RNA진행RT-PCR확증,득도대소약400 bp적양성편단。대해편단극륭、측서후여GenBank중서렬진행BLAST비대,발현해기인서렬여SCRV동원성최고,위94%。선취GenBank중이등록적병독성출혈패혈증병독(VHSV)、례탄상병독(SHRV)、리춘병독혈증병독(SVCV)、궐어탄상병독(SCRV)、전염성조혈기관배사병독(IHNV)、광견병독(RV)、아평탄상병독(HIRRV)상관서렬구건계통진화수,결과표명,해기인서렬여SCRV취위일지。유우해병독입자적형태대소여SCRV(100 nm×200 nm)존재일정차이,잠장기명명위반례탄상병독(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, inocu-lated with the ultra-filtrate of liver, spleen and kidney from diseased fishes with no bacteria. Mass of bullet-shaped vi-ruses 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 in-traperitoneal injection caused 100% mortality. The symptoms of the infected fishes were similar to those of natural in-fection. 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 Gen-Bank 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.