Ding Zhengfeng, Xia Siyao, Zhao Ziming, Xia Aijun, Shen Meifang, Tang Jianqing, Xue Hui, Geng Xuebing, Yuan Sheng
Freshwater Fisheries Research Institute of Jiangsu Province, 79 Chating East Street, Nanjing 210017, China.
College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China.
J Virol Methods. 2014 Sep;206:76-83. doi: 10.1016/j.jviromet.2014.05.011. Epub 2014 May 27.
Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen in the commercially exploited fish, Prussian carp (Carassius auratus gibelio), which has caused huge economic loss in China and appears to be spreading worldwide. In this article, CyHV-2 infection of Prussian carp was confirmed for the first time by polymerase chain reaction (PCR), which gave positive results from the tissue samples dissected from moribund fish including kidney, spleen, liver, and gill. Histological examination showed systemic inflammatory reactions in the infected tissues, with infiltration of hemocytes, hypertrophied nuclei, marginal chromatin and karyorrhexis, epithelial cell shedding, vacuolar degeneration and focal necrosis. Tissue alterations were also evaluated semi-quantitatively by the degree of tissue change. The values of degree of tissue change determined for kidney, spleen, liver, and gill were significantly greater than respective controls and kidney was the most severely damaged organ, with highest degree of tissue change value. In addition, a fluorescence in situ hybridization (FISH) based on oligonucleotide probes to detect the pathogen directly in the tissue, allowing pathogen-lesion correlation, was established. With the advantages of better tissue penetration, potentially more specific and stable, three oligonucleotide probes were designed. Positive reactions to the probes with intense green fluorescence were observed within the infected tissues where PCR and H&E analysis had suggested previously the presence of the virus within these lesions. The probes did not hybridize with host tissues of uninfected fish, nor did they cross-react with 3 other virus samples tested. The current research could facilitate the study of CyHV-2 infection mechanism in Prussian carp, and enhance the early diagnosis of the novel virus.
鲤疱疹病毒2型（CyHV-2）是商业养殖鱼类银鲫（Carassius auratus gibelio）中出现的一种病原体，它在中国已造成巨大经济损失，且似乎正在全球范围内传播。在本文中，首次通过聚合酶链反应（PCR）证实了银鲫感染CyHV-2，从濒死鱼解剖得到的组织样本（包括肾脏、脾脏、肝脏和鳃）中PCR检测呈阳性。组织学检查显示，感染组织出现全身性炎症反应，有血细胞浸润、细胞核肥大、核边缘染色质和核碎裂、上皮细胞脱落、空泡变性和局灶性坏死。还通过组织变化程度对组织改变进行了半定量评估。确定的肾脏、脾脏、肝脏和鳃的组织变化程度值显著高于各自的对照组，肾脏是受损最严重的器官，组织变化程度值最高。此外，建立了一种基于寡核苷酸探针的荧光原位杂交（FISH）方法，可直接在组织中检测病原体，实现病原体与病变的关联。由于具有更好的组织穿透力、可能更特异和稳定的优点，设计了三种寡核苷酸探针。在PCR和苏木精-伊红（H&E）分析先前提示存在病毒的感染组织内，观察到对探针有强烈绿色荧光的阳性反应。这些探针未与未感染鱼的宿主组织杂交，也未与其他3种测试病毒样本发生交叉反应。当前的研究有助于对银鲫中CyHV-2感染机制的研究，并加强对这种新型病毒的早期诊断。
