稳定表达eGFP-戊型肝炎病毒复制子的建立及体内外特征

Development and Characterization of a Stable eGFP-hepatitis E Virus Replicon in vitro and in vivo

戊型肝炎病毒(HEV)是急性病毒性肝炎的主要病原体.为了寻找有效的HEV研究工具,利用反向遗传学构建了携带绿色荧光蛋白(eGFP)的重组HEV感染性克隆——eGFP-HEV.eGFP-HEV转染Huh7.5.1细胞后,在荧光显微镜下能够直接观察到荧光信号,可以实时追踪HEV的复制情况,将eGFP-HEV的子代病毒粒子接种细胞,利用免疫荧光和qRT-PCR验证了其传染性.eGFP-HEV细胞培养系统可以通过直接观察绿色荧光信号来评价药物的抗病毒效果,为HEV药物筛选提供了重要工具.此外,在接种eGFP-HEV的BALB/c裸鼠的胆汁和粪便中也可追踪到eGFP-HEV的绿色荧光信号.值得注意的是,感染小鼠肝脏中表达的HEV抗原量与eGFP的荧光强度高度匹配,可以利用eGFP的荧光信号代表HEV的病毒滴度.携带绿色荧光蛋白的eGFP-HEV感染性克隆在体内、外的成功表达将加快HEV的研究,有利于抗HEV药物或疫苗的研发.

Hepatitis E virus(HEV)is the main pathogen of acute viral hepatitis.To explore an efficient tool for HEV study,recombinant HEV,eGFP-HEV,expressing enhanced green fluorescence protein(eGFP)followed the full-length HEV ORF2 gene was generated through reverse genetics.The green fluorescence signals can be directly viewed under a fluorescence microscope to trace the replication of HEV after transfection of eGFP-HEV in Huh7.5.1 cells.The infectivity of eGFP-HEV was verified by inoculating progeny virion into na ve fresh cells.The cell culture system of eGFP-HEV can be used to evaluate the antiviral effects by directly viewing the green fluorescence signals,which supply a tool for HEV drug development.Furthermore,the signals were traceable in the bile and stool of BALB/c nude mice inoculated with eGFP-HEV.Remarkably,the HEV antigens expressed in the livers of infected mice were perfectly matched with the fluorescent intensity of eGFP,which indicated that the fluorescence signals from eGFP represent the viral titer of HEV.Establishing eGFP-HEV with visible green fluorescence signals in vitro and in vivo will accelerate the studies on HEV and benefit the development of anti-HEV drugs or vaccines.