Stiftung Tierärztliche Hochschule Hannover (TiHo)

Establishment of a plasmid-based reverse genetics system for the cell culture-adapted hepatitis E virus genotype 3c strain 47832c

Scholz, Johannes; Bächlein, Christine GND; Gadicherla, Ashish K.; Falkenhagen, Alexander; Tausch, Simon H. ORCID; Johne, Reimar

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 in vitro 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.

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Scholz, Johannes / Bächlein, Christine / Gadicherla, Ashish / et al: Establishment of a plasmid-based reverse genetics system for the cell culture-adapted hepatitis E virus genotype 3c strain 47832c. 2020.

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