广东地区猪群新发塞内卡病毒流行株的分离鉴定及遗传进化分析

Isolation, Identification and Genetic Evolution Analysis of New Epidemic Strains of Senecavirus A in Guangdong Province

【目的】检测广东两猪场新发的鼻镜与蹄冠水疱病病原,获得新发塞内卡病毒(Senecavirus A,SVA)流行株,为后续研究提供材料。【方法】应用SVA RT-PCR、荧光PCR等方法对采集的病料进行初步筛查,再将水泡皮进行处理接种于ST细胞、BHK-21细胞上,用SVA特异性引物对细胞上清液进行扩增,测序并分析序列,采用蔗糖浓度梯度方法纯化病毒进行电子显微镜形态观察,并对病毒分离株的TCID50进行测定。【结果】经SVA RT-PCR与荧光PCR方法初步诊断该猪群为SVA感染;细胞接毒培养发现,自第6代起,细胞出现典型CPE,电镜观察可见25 nm左右的病毒颗粒,扩增序列提交NCBI Blastn比对鉴定为SVA;测得毒株SVA CH-GDBL1-2016的TCID50为106.8,毒株SVA CH-GDBL2-2016的TCID50为106.7。【结论】SVACH-GDBL1-2016和SVA CH-GDBL2-2016可为后续塞内卡病毒病的诊断和疫苗研制提供材料基础。

【Objective】The pathogen of blistering disease with nasal and coronet in two swine farms in Guangdong province was detected,and new strains of Senecavirus A(SVA)were obtained,which provided materials for subsequent research.【Method】RT-PCR and real-time RT-PCR were used to screen the clinical samples.The clinical samples were isolated,treated and cultured with ST cells and BHK-21 cells.The virus was purified by sucrose density gradient centrifugation method and observed by electron microscopy.The specific primers of SVA were designed to amplify the cell supernatant,and sequenced for sequence analysis.The TCID50 of the virus isolated was determined.【Results】The swinery were diagnosed as SVA by RT-PCR and real-time RT-PCR.It was found that the cultured cells had CPE from the 6th generation.The virion observed by electron microscopy was about 25 nm.The amplified sequences were submitted to the NCBI Blastn,which were identified as SVA.The TCID50 result of the strain SVA CH-GDBL1-2016 was 106.8 and the TCID50 result of the strain SVA CH-GDBL2-2016 was 106.7.【Conclusion】The strains SVA CH-GDBL1-2016 and SVA CH-GDBL2-2016 provide a material basis for the subsequent diagnosis and vaccine development of Senecavirus disease.