国家重点研发计划 (2018YFD0900501)；国家自然科学基金 (31872589)；广东省现代农业产业技术创新团队专项 (2019KJ140, 2019KJ141)；广东省海洋经济发展 (六大产业)专项基金项目“海洋源虹彩病毒高效疫苗创制与应用”；中国水产科学研究院中央级公益性科研院所基本科研业务费专项 (2019SJ-XT4)；广州市科技计划项目 (202002030045)
为探讨大口黑鲈蛙病毒 (LMBV) 的分子流行规律及在感染后的组织病理变化，实验对2019—2021年采集的723份患病大口黑鲈样品进行荧光定量PCR (qPCR)检测。结果显示，LMBV的阳性率为63.62%，挑选阳性样品接种鳜脑细胞 (Chinese perch brain cells, CPB)，共获得93株LMBV。通过对分离株MCP、ATP酶、DNA聚合酶和甲基转移酶基因进行PCR扩增与测序分析，发现上述基因在LMBV分离株中均保守，其中MCP、ATP酶基因一致性均为100.0%、甲基转移酶基因一致性为99.7%~100.0%、DNA聚合酶基因一致性为99.8%~100.0%。选取1株LMBV毒株感染健康大口黑鲈，采用qPCR方法对不同组织中的病毒载量进行检测，结果显示，大口黑鲈蛙病毒在心脏、肝脏、脾脏、肾脏、胃、肠和脑等组织中均有分布，人工感染LMBV后的前5天病毒载量逐步升高；感染后第5天，心脏中病毒载量最高 (9.5×105个/ mg拷贝)，脑组织中病毒载量最低 (2.9×102个/ mg拷贝)。组织病理结果表明，LMBV可导致大口黑鲈多种组织坏死，其中肝脏、肾脏和心脏病变较为严重，典型病理特征包括肝细胞排列紊乱、肝窦扩张、核质疏散；肾组织细胞疏散、巨噬细胞破裂；心室外膜纤维坏死等。实验结果可为大口黑鲈蛙病毒病防控提供参考。
Micropterus salmoides is a high-quality freshwater fish among the main aquaculture species in my country. In recent years, diseases of M. salmoides have occurred frequently, which has caused a great impact on my country's aquaculture industry. Largemouth bass ranavirus (LMBV) is a pathogen that seriously harms the M. salmoides farming industry. In order to explore the pathological changes of M. salmoides after infection with LMBV, and the relationship between the dynamic changes of virus content in the body and the occurrence of diseaseases. The typical symptom of diseaseased fish is muscle necrosis, commonly known as rotting. In this study, 723 diseased M. salmoides samples collected from 2019 to 2021 in Guangdong Province and surrounding areas were classified and sorted, and their liver, spleen and kidney tissues were collected for quantitative PCR detection.. The results showed that the rate of positive LMBV was 63.62%. In addition, the incidence and detection rate are higher in the summer high temperature season. LMBV positive samples were selected to inoculate CPB cells, and a total of 93 strains of LMBV were obtained. Through PCR amplification and sequencing analysis of the isolates MCP, ATPase, DNA polymerase and methyltransferase, the results showed that these genes are highly conserved in LMBV isolates. The identity of the MCP and ATPase gene is 100.0%, and the methyltransferase gene identity is 99.7%-100.0%, and the DNA polymerase gene identity is 99.8%-100.0%, indicating that the variation between LMBV strains is minimal. The representative strain was selected to inject 100 μL 3.72×106 TCID50/mL LMBV into the pectoral fins of M. salmoides to artificially infect the fish with LMBV. The samples showed similar symptoms to those infected with LMBV in the natural state, including darkening of the fish body, surface ulceration, and liver atrophy, etc. Four fish were randomly selected at 0 h 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d after infection, and liver, spleen, kidney, stomach, intestine, brain, heart and gill tissues were taken to make paraffin sections with HE staining for histopathological changes analysis. qPCR was used to detect the tissue distribution and kinetics of the virus. The results showed that the viral load of LMBV gradually increaseased within 5 days after infection, and the virus was distributed in the heart, liver, spleen, kidney, stomach, intestine, brain and other tissues, and the highest viral load in the heart was 9.5×105 copies/mg. The minimum viral load in brain tissue was 2.9×102 copies/mg. The histopathological results after virus infection showed that LMBV can cause a variety of tissue necrosis of M. salmoides, among which liver, kidney and heart diseases become more serious. Typical pathological features include disorder of liver cell arrangement, dilatation of liver sinusoids, nucleus evacuation, renal tissue cell evacuation, Macrophage rupture, necrosis of ventricular membrane fibers, etc.
YANG Zhanzhan, LIN Qiang, FU Xiaozhe, LUO Xia, LIU Lihui, LIANG Hongru, NIU Yinjie, ZUO Shaozhi, ZHANG Xiaoting, LI Ningqiu. Molecular epidemiology and histopathological analysis of largemouth bass ranavirus[J]. Journal of Fisheries of China,2022,46(6):1063~1073复制