Porcine epidemic diarrhea virus(PEDV)infection causes acute watery diarrhea in neonatal piglets,leading to substantial economic losses within the pig farming industry.This study demonstrates that clofazimine(CFZ)signi...Porcine epidemic diarrhea virus(PEDV)infection causes acute watery diarrhea in neonatal piglets,leading to substantial economic losses within the pig farming industry.This study demonstrates that clofazimine(CFZ)significantly inhibits PEDV replication in a dose-dependent manner in vitro,with negligible cytotoxicity.Findings from our time-of-addition assays indicate that CFZ effectively disrupts multiple stages of the viral infection cycle.Using a CoV-RdRp-Gluc reporter system,we evaluated the potency of CFZ against PEDV RNA-dependent RNA polymerase(RdRp),and determined a low IC50 value of 0.1364μM.Molecular docking studies further confirmed that CFZ has high binding affinity at the active sites of the spike protein and RdRp protein in PEDV.Transcriptome analysis of Vero E6 cells,with and without CFZ treatment,revealed a significant change in transcriptional activity at 8 h postinfection(hpi).Moreover,the simultaneous application of CFZ and nucleoside analogs showed enhanced the anti-PEDV effect of CFZ in vitro.Our study underscores the potential of CFZ as a viable therapeutic agent against PEDV.展开更多
Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02...Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02/LZ35666 shared high sequence homology with GII-4 NVs.Nucleotide homologies of RdRp region and encoded capsid protein region were 90.4%-98.6% and 89.8%-95.7%,respectively,while amino acid homology of capsid protein region was 94.4%-97.4%.The analysis of GDD motif in RdRp region indicated this GDD motif of Lanzhou strain differed from those of the GII-4 predominant epidemic strains.Lanzhou strain formed an independent branch in GII-4 cluster in the phylogenetic tree based on nucleotide sequence of RdRp region and amino acid sequence of capsid protein.Sequence alignment revealed a mutation at the fourth key site of the receptor-binding interface in the strains isolated after 2002 compared with those of previous strains suggesting a possible change of binding pattern to HBGAs receptors.展开更多
目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体...目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体中,并在其3′端插入EGFP报告基因,构建融合表达质粒pcDNA3.0-RdRp-EGFP,脂质体法转染PLC/PRF/5、A549和HepG2细胞,荧光显微镜观察报告基因的表达,RT-nPCR检测RdRp基因mRNA的转录情况,Westernblot检测RdRp蛋白的表达。结果重组表达质粒经酶切鉴定和测序证实构建正确;RdRp基因和蛋白在A549和HepG2细胞中可高效、特异性表达。结论成功构建了RdRp基因真核表达质粒,并在A549和HepG2培养细胞中大量表达,为进一步研究RdRp在HEV复制过程中的功能奠定了基础。展开更多
基金supported by a grant from the National Key Research and Development Plans of China(No.2021YFD1800401)to Zhe Wangthe National Natural Science Foundation of China(No.32070128)to Zhe Wang+5 种基金the Shanghai Biomedical Science and Technology Support Special Project(No.21S11900200)to Zhe Wangthe National Natural Science Foundation of China(No.31472211)to ZhiBiao Yangthe Natural Science Foundation of Shanghai(No.21ZR1433900)to Zhibiao Yangthe National Key Research and Development Program of China(No.2022YFD1800805)to Yanjun Zhouthe Pinduoduo-China Agricultural University Research Fund(PC2024A01003)to Xiangfeng Wangthe Biomedicine and Technology Supporting Project of the Shanghai Science and Technology Innovation Plan(Grant No.22S11902200,China)to Tao Sun.
文摘Porcine epidemic diarrhea virus(PEDV)infection causes acute watery diarrhea in neonatal piglets,leading to substantial economic losses within the pig farming industry.This study demonstrates that clofazimine(CFZ)significantly inhibits PEDV replication in a dose-dependent manner in vitro,with negligible cytotoxicity.Findings from our time-of-addition assays indicate that CFZ effectively disrupts multiple stages of the viral infection cycle.Using a CoV-RdRp-Gluc reporter system,we evaluated the potency of CFZ against PEDV RNA-dependent RNA polymerase(RdRp),and determined a low IC50 value of 0.1364μM.Molecular docking studies further confirmed that CFZ has high binding affinity at the active sites of the spike protein and RdRp protein in PEDV.Transcriptome analysis of Vero E6 cells,with and without CFZ treatment,revealed a significant change in transcriptional activity at 8 h postinfection(hpi).Moreover,the simultaneous application of CFZ and nucleoside analogs showed enhanced the anti-PEDV effect of CFZ in vitro.Our study underscores the potential of CFZ as a viable therapeutic agent against PEDV.
文摘Sequence analysis of a new norovirus(NV) isolated from Lanzou city of China was performed based on partial sequence of RNA dependent RNA polymerase(RdRp) and complete capsid protein(VP1) gene.The isolated strain CHN02/LZ35666 shared high sequence homology with GII-4 NVs.Nucleotide homologies of RdRp region and encoded capsid protein region were 90.4%-98.6% and 89.8%-95.7%,respectively,while amino acid homology of capsid protein region was 94.4%-97.4%.The analysis of GDD motif in RdRp region indicated this GDD motif of Lanzhou strain differed from those of the GII-4 predominant epidemic strains.Lanzhou strain formed an independent branch in GII-4 cluster in the phylogenetic tree based on nucleotide sequence of RdRp region and amino acid sequence of capsid protein.Sequence alignment revealed a mutation at the fourth key site of the receptor-binding interface in the strains isolated after 2002 compared with those of previous strains suggesting a possible change of binding pattern to HBGAs receptors.
文摘目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体中,并在其3′端插入EGFP报告基因,构建融合表达质粒pcDNA3.0-RdRp-EGFP,脂质体法转染PLC/PRF/5、A549和HepG2细胞,荧光显微镜观察报告基因的表达,RT-nPCR检测RdRp基因mRNA的转录情况,Westernblot检测RdRp蛋白的表达。结果重组表达质粒经酶切鉴定和测序证实构建正确;RdRp基因和蛋白在A549和HepG2细胞中可高效、特异性表达。结论成功构建了RdRp基因真核表达质粒,并在A549和HepG2培养细胞中大量表达,为进一步研究RdRp在HEV复制过程中的功能奠定了基础。
