四川地区养殖鲫鲤疱疹病毒Ⅱ型的鉴定及病理学研究
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S941.41

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四川省留学回国人员科技活动项目择优资助;四川省教育厅基础研究项目(17ZB0345)


Identification and pathological study of Cyprinid herpesvirus II in Sichuan
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    摘要:

    为探究疾病病因和流行特点,对患病鲫进行剖检、病理学观察、分子生物学检测及人工感染实验。结果显示,患病鲫临床表现为离群独游、浮头、全身发黑及体表出血等。剖检发现鳃、肝脏、肾脏等器官出血、肿胀和坏死,鳔点状出血。组织病理观察显示,鳃呼吸上皮细胞肿胀、坏死及脱落;肾脏局灶性坏死;脾脏组织广泛变性坏死,造血系统崩解;肝脏发生空泡变性和坏死。透射电镜观察到脾脏和肾脏中存在4种大小不同的病毒颗粒,分别为DNA病毒内核、空衣壳、实心衣壳和有包膜的成熟病毒粒子。未成熟的病毒粒子存在于细胞核,成熟的病毒粒子存在于细胞质。电镜下可见鲤疱疹病毒Ⅱ型(CyHV-2)在细胞核内完成核酸复制和核衣壳装配,囊膜蛋白在出核后获得。细胞核萎缩、核染色质边缘化,线粒体肿胀、坏死、嵴断裂,细胞质空泡化。取患病鲫组织匀浆滤菌后接种到鲤上皮瘤细胞系(epithelioma papulosum cyprinid cell line,EPC)和胖头鲤肌肉细胞系(fathead minnow cell line, FHM),盲传3代后未见细胞病变。通过腹腔注射患病鲫组织匀浆进行人工感染实验,实验组鲫表现出与自然感染鲫相同的临床症状,对照组鲫正常,实验组累积死亡率为80%。用PCR方法检测CyHV-2的解旋酶基因片段,扩增出预期大小相符的目的条带。利用邻近法对该病毒的解旋酶基因进行系统发育分析,结果显示与CyHV-2(YZ-01)的同源性为99%。以上实验结果证实了CyHV-2是导致这次疾病暴发的原因。

    Abstract:

    In October 2018, crucian carp (Carassius auratus) in a farm located in Zigong, Sichuan Province, developed a communicable disease with a mortality rate of 80%. In order to explore the cause of this disease, sick fish were subjected to necropsy, pathological observation, molecular biological examination and artificial infection test. The clinical manifestations of diseased fish included leaving the shoal of fish to swim alone; staying on the water surface; bodies blackened and body surface had haemorrhages. The necropsy showed haemorrhage, swelling and necrosis of organs such as gills, liver and kidney; swimming bladder had a lot of blutene chloaide. Histopathological changes showed that the respiratory epithelial cells of gill were swollen, necrosis and sloughting. The kidneys showed focal necrosis. The spleen tissue was extensively degenerated and necrotic, and the hematopoietic system collapsed. Vacuolar degeneration and necrosis occurred in the liver. Transmission electron microscopy showed that there were four different sizes of virus particles in the spleen and kidney, which were virus DNA core, empty viral capsid, solid capsid and enveloped mature virions. It can be seen that immature virions are present in the nucleus, and mature virions are present in the cytoplasm. Cyprinid herpesvirus Ⅱ (CyHV-2) completed nucleic acid replication and nucleocapsid assembly in the nucleus, and the envelope protein was obtained after transmembrane membrane. After filtering the diseased fish tissue homogenate, it was inoculated into EPC and FHM cell lines, and there was no cytopathic effect after 3 generations of blind transmission. The helicase gene fragment of CyHV-2 was amplified by PCR, and a positive band appeared. The artificial infection test was carried out by intraperitoneal injection of tissue homogenate. The test group showed the same clinical symptoms as the natural infection fish and the control group was normal. The cumulative mortality rate of the test group was 80%. Phylogenetic analysis of helicase gene by the neighbor-joining method revealed that the homology with CyHV-2 (YZ-01) was 99%. The results demonstrated that CyHV-2 contributed to this disease outbreak.

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周瑶佳,田思璐,许佳雪,李尹麒,杨悦,耿毅,黄小丽,陈德芳,汪开毓,欧阳萍.四川地区养殖鲫鲤疱疹病毒Ⅱ型的鉴定及病理学研究[J].水产学报,2020,44(9):1397~1407

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  • 收稿日期:2019-07-11
  • 最后修改日期:2019-10-22
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  • 在线发布日期: 2020-09-27
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