Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c
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 represent...
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doaj-9c98df9ad7d94e5aa7f3f87c80cbc11e2020-11-25T03:32:29ZengMDPI AGPathogens2076-08172020-02-019315710.3390/pathogens9030157pathogens9030157Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832cJohannes Scholz0Christine Bächlein1Ashish K. Gadicherla2Alexander Falkenhagen3Simon H. Tausch4Reimar Johne5Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, GermanyInstitute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, GermanyDepartment Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, GermanyDepartment Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, GermanyDepartment Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, GermanyDepartment Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, GermanyThe 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.https://www.mdpi.com/2076-0817/9/3/157hepatitis e virusgenotype 3ccell culturereverse genetics systemsite-directed mutagenesis |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Johannes Scholz Christine Bächlein Ashish K. Gadicherla Alexander Falkenhagen Simon H. Tausch Reimar Johne |
spellingShingle |
Johannes Scholz Christine Bächlein Ashish K. Gadicherla Alexander Falkenhagen Simon H. Tausch Reimar Johne Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c Pathogens hepatitis e virus genotype 3c cell culture reverse genetics system site-directed mutagenesis |
author_facet |
Johannes Scholz Christine Bächlein Ashish K. Gadicherla Alexander Falkenhagen Simon H. Tausch Reimar Johne |
author_sort |
Johannes Scholz |
title |
Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c |
title_short |
Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c |
title_full |
Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c |
title_fullStr |
Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c |
title_full_unstemmed |
Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c |
title_sort |
establishment of a plasmid-based reverse genetics system for the cell culture-adapted hepatitis e virus genotype 3c strain 47832c |
publisher |
MDPI AG |
series |
Pathogens |
issn |
2076-0817 |
publishDate |
2020-02-01 |
description |
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. |
topic |
hepatitis e virus genotype 3c cell culture reverse genetics system site-directed mutagenesis |
url |
https://www.mdpi.com/2076-0817/9/3/157 |
work_keys_str_mv |
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