Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c

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.

The Translated Amino Acid Sequence of an Insertion in the Hepatitis E Virus Strain 47832c Genome, But Not the RNA Sequence, Is Essential for Efficient Cell Culture Replication

“…The HEV strain 47832c is a GT3c strain, which was originally isolated from a chronically infected kidney transplant patient from Germany [ 23 ]. The cDNA clone p47832mc (Genbank accession number MN756606) contains the complete genome of strain 47832c under control of a T7 RNA polymerase promotor [ 32 ] and can be used for the generation of replicating virus by transfection into BSR T7/5 cells.…”

“…The mutants were generated by modification of plasmid p47832mc using methods as previously described [ 32 ]. Briefly, DNA fragments containing the desired deletions, sequence substitutions or point mutations (schematic view in Figure 1 and details in Supplementary Table S1 ) were synthesized by commercial providers (Integrated DNA Technologies, Coralville, IA, USA or GenScript Biotech B.V., Leiden, The Netherlands).…”

“…Either the cloned fragments or the synthesized fragments directly were digested with specific restriction endonucleases (Thermo Fisher Scientific, Waltham, MA, USA or New England Biolabs (NEB), Schwalbach, Germany) and the resulting fragments were substituted with the respective fragments of the digested plasmid p47832mc. The sequences of the resulting genomic plasmids were validated by NGS, as described previously [ 32 ]. In addition, the substituted regions of the plasmids were validated using Sanger sequencing (Eurofins Genomics Germany GmbH, Ebersberg, Germany).…”

“…In the presented work, the significance of the insertion in the HVR of strain 47832c for its replication in cell culture was determined and functionally important parts of the insertion were analyzed. Using a recently established reverse genetics system for this virus strain [ 32 ], several mutants carrying deletions, sequence substitutions or point mutations in the respective region were generated and tested for their growth in cell culture. By this, it is shown that the presence of the original translated amino acid sequence of the insertion, but not its nucleotide sequence, is crucial for efficient replication of the strain in cell culture.…”

Hepatitis E Virus Infection: Circulation, Molecular Epidemiology, and Impact on Global Health

“…Reverse genetics models based on infectious cDNA clones have been described for several genotypes. The most commonly used are the Sar-55-related genotype 1 clone [ 68 ], the genotype 3a and 3c Kernow-C1- [ 69 ], and 47832-related [ 70 ] clones, respectively, both of which contain insertions in the HVR, and the genotype 4 TW6196 clone [ 71 ]. A recent presentation of a novel in vitro method to produce high viral titers, allowing study of the full HEV replication cycle in cell culture, has additionally created confidence that we may overcome our limited understanding of HEV pathophysiology [ 72 ].…”