The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into...The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.展开更多
Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at pos...Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at position 100 of VP1.The amino acids isoleucine,alanine,serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis,and viable viruses were rescued.Although all mutants and wild-type viruses display different plaque morphologies,they replicate comparably in BHK-21 cells.The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice.The results showed that the isoleucine(T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality,lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus.These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection,and its mutation alters the pathogenicity of viral infection in mice.展开更多
基金supported by the Key Project of National Natural Science Foundation of China(30530550)
文摘The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.
文摘【目的】利用网络药理学和分子对接技术发现莪术醇抗脑心肌炎病毒(Encephalomyocarditis virus,EMCV)的作用靶点及机制。【方法】利用PharmMapper、GeneCards数据库获得莪术醇抗EMCV的相关靶点;通过Cytoscape 3.7.2软件、STRING和DAVID数据库构建靶蛋白互作(PPI)网络并筛选关键靶点,对靶点进行GO功能和KEGG通路富集分析,并构建莪术醇抗EMCV靶点-通路网络;通过AutoDock Vina 1.1.2分析莪术醇与靶蛋白的结合能及结合模式。【结果】网络药理学分析结果显示,莪术醇抗EMCV的潜在靶点有9个,其中丝裂原活化激酶14(mitogen-activated protein kinase 14,MAPK14)、信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)、白蛋白(albumin,ALB)、白细胞介素2(interleukin 2,IL2)可能是莪术醇抗EMCV的核心靶点,所得靶点参与C型凝集素受体信号通路、神经营养素信号通路和JAK-STAT信号通路等代谢通路,功能涉及调节炎症反应细胞因子的产生、蛋白激酶活性和药物结合等;分子对接结果显示,4种核心靶蛋白与莪术醇之间存在较强的结合能,均存在疏水形式的结合,其中ALB、STAT1和IL2与莪术醇之间还存在氢键结合。【结论】本研究结果表明,MAPK14、STAT1、ALB和IL2是莪术醇发挥抗EMCV作用的潜在靶点,本研究为莪术醇作为抗EMCV药物的研发提供理论依据和线索。
基金supported by the National Natural Science Foundation of China (Grant No. 30530550)the Program for Innovative Research Team in University of China (Grant No. IRT0866)
文摘Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at position 100 of VP1.The amino acids isoleucine,alanine,serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis,and viable viruses were rescued.Although all mutants and wild-type viruses display different plaque morphologies,they replicate comparably in BHK-21 cells.The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice.The results showed that the isoleucine(T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality,lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus.These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection,and its mutation alters the pathogenicity of viral infection in mice.
