感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究

Research on Histopathological Changes and Virus Distribution Situation of Mirror Carps Infected with Carp Edema Virus

【目的】研究感染鲤浮肿病毒(Carp edema virus,CEV)镜鲤的组织病理特征及CEV在各组织中的分布规律,以期为鲤浮肿病的诊断和防控提供参考。【方法】利用HE染色观察发病镜鲤心脏、肝脏、皮肤、脾脏、肾脏、脑、鳃组织的病理变化情况;用免疫组织化学法检测CEV在镜鲤各组织的分布和蛋白表达情况;利用CEV P4a基因保守区域设计3条探针,于5′-端标记FAM进行原位杂交,检测CEV在镜鲤各组织的核酸分布情况;实时荧光定量PCR检测感染镜鲤各组织的病毒载量。【结果】HE染色观察发现,患病镜鲤的鳃丝肿胀、充血,鳃丝间有脱落堆积的红细胞和炎性细胞,肝脏细胞深染萎缩,胆小管出血,脾脏组织出现明显间隙并伴有出血,肾脏组织充血明显,肾小管有明显闭合现象。免疫组织化学结果显示,在患病鱼的鳃和肝脏组织中大量表达CEV P4a蛋白,在其他组织中也有少量表达。原位杂交结果显示,CEV核酸大量存在于鳃组织中,在心脏、脾脏和肾脏中均有少量阳性信号。实时荧光定量PCR结果显示,鳃组织病毒载量极显著高于心脏、肝脏、皮肤、脾脏、肾脏、脑组织(P<0.01)。【结论】感染CEV后,镜鲤内脏组织出现不同程度充血、出血,病毒主要分布在鳃,说明鳃是CEV增殖的最主要靶器官。

【Objective】This study was aimed to study the histopathological characteristics of mirror carps infected with Carp edema virus(CEV)and the distribution of CEV in various tissues,in order to provide references for the diagnosis,prevention and control of Carp edema virus disease(CEVD).【Method】The pathological changes of heart,liver,skin,spleen,kidney,brain and gill tissues of infected mirror carps were observed by HE staining.The immunohistochemistry was used to detect the distribution and protein expression of CEV in the tissues of mirror carps.Three probes were designed in the conserved region of CEV P4a gene and labeled at the 5′end FAM for in situ hybridization to detect the nucleic acid distribution of CEV in the tissues of mirror carps.Real-time quantitative PCR was used to detect the viral load in each tissue of diseased mirror carps.【Result】HE staining analysis showed that the gills of the diseased mirror carps were swollen and hyperemic,and there were red blood cells and inflammatory cells between the gills.The liver cells were deeply stained and atrophied,and the bile duct was bleeding.There was obvious space and hemorrhage in spleen tissue.The renal tissue hyperemia was obvious and the renal tubules were obviously closed.The results of immunohistochemistry showed that the CEV P4a protein was highly expressed in gill and liver tissues of the diseased fish,and less expressed in other tissues.The results of in situ hybridization showed that CEV nucleic acids were abundant in gill tissue,and a small amount of positive signals were found in heart,spleen and kidney.Real-time quantitative PCR assay indicated that the viral load of gill was extremly significantly higher than that in myocardium,liver,skin,spleen,kidney and brain tissues(P<0.01).【Conclusion】After infection with CEV,visceral tissues of mirror carps showed varying degrees of congestion and bleeding,the virus was mainly distributed in gill,indicating that gill was the main target organ for CEV proliferation.