Scholz Johannes, Bächlein Christine, Gadicherla Ashish K, Falkenhagen Alexander, Tausch Simon H, Johne Reimar
Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany.
Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany.
Pathogens. 2020 Feb 25;9(3):157. doi: 10.3390/pathogens9030157.
The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.
戊型肝炎病毒（HEV）可导致人类急性和慢性肝炎。由于缺乏广泛适用的、高效的细胞培养系统以及用于HEV定点诱变的工具，对HEV复制的研究受到了阻碍。细胞培养适应型3c基因型毒株47832c是欧洲主要检测到的典型基因型，此前已用于多项基础和应用研究。在此，针对该毒株开发了一种基于质粒的反向遗传学系统，该系统无需RNA转录即可高效拯救感染性病毒。将表达T7 RNA聚合酶的BSR/T7细胞与一个编码全长病毒基因组的质粒以及两个编码痘苗病毒封端酶的辅助质粒共转染，可产生感染性HEV，其可在A549/D3细胞上连续传代。亲代病毒和重组病毒表现出相似的复制动力学。可成功地将产生额外限制性酶切位点的单点突变引入子代病毒的病毒基因组，表明该系统适用于定点诱变。该系统是首个基于质粒的HEV反向遗传学系统，也是首个针对HEV 3c基因型的反向遗传学系统，因此应广泛应用于HEV的基础和应用研究。
