Abstract
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.
Highlights
Hepatitis E is a human disease which is highly prevalent worldwide with more than 20 million infections and over three million cases each year [1]
The disease is caused by infection with the hepatitis E virus (HEV), which is classified into the genus
HEV is fecally excreted as a nonenveloped particle, but enveloped particles have been identified in serum and cell culture supernatants [5]
Summary
Hepatitis E is a human disease which is highly prevalent worldwide with more than 20 million infections and over three million cases each year [1]. The disease is caused by infection with the hepatitis E virus (HEV), which is classified into the genus. To show the suitability of the system for site-directed mutagenesis, a silent point mutation resulting. To show the suitability of thesite system for site‐directed mutagenesis, a silent point mutation in a newly generated. The position of the nucleotide substitution within the insertion in ORF1 of the genome of the of nucleotide substitution the insertion in ORF1 of the in plasmid is shown in p47832 Figure EcoRI.
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