{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T20:10:58Z","timestamp":1762459858494,"version":"build-2065373602"},"reference-count":28,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2020,2,25]],"date-time":"2020-02-25T00:00:00Z","timestamp":1582588800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"German Federal Institute for Risk Assessment","award":["GOHI 1322-686"],"award-info":[{"award-number":["GOHI 1322-686"]}]},{"DOI":"10.13039\/501100003107","name":"Bundesministerium f\u00fcr Gesundheit","doi-asserted-by":"publisher","award":["CHED 321-4471-02\/157"],"award-info":[{"award-number":["CHED 321-4471-02\/157"]}],"id":[{"id":"10.13039\/501100003107","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/pathogens9030157","type":"journal-article","created":{"date-parts":[[2020,2,26]],"date-time":"2020-02-26T04:18:29Z","timestamp":1582690709000},"page":"157","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c"],"prefix":"10.3390","volume":"9","author":[{"given":"Johannes","family":"Scholz","sequence":"first","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christine","family":"B\u00e4chlein","sequence":"additional","affiliation":[{"name":"Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashish K.","family":"Gadicherla","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander","family":"Falkenhagen","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6874-233X","authenticated-orcid":false,"given":"Simon H.","family":"Tausch","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reimar","family":"Johne","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,2,25]]},"reference":[{"key":"ref_1","unstructured":"WHO (2019, July 08). Fact sheet Hepatitis E. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/hepatitis-e."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1111\/jvh.12445","article-title":"Hepatitis E: An emerging global disease\u2014from discovery towards control and cure","volume":"23","author":"Khuroo","year":"2016","journal-title":"J. Viral Hep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"30561","DOI":"10.2807\/1560-7917.ES.2017.22.26.30561","article-title":"Hepatitis E virus infection in Europe: Surveillance and descriptive epidemiology of confirmed cases, 2005 to 2015","volume":"22","author":"Aspinall","year":"2017","journal-title":"Eurosurveillance"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2645","DOI":"10.1099\/jgv.0.000940","article-title":"ICTV Report Consortium. ICTV Virus Taxonomy Profile: Hepeviridae","volume":"98","author":"Purdy","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e00822-17","DOI":"10.1128\/JVI.00822-17","article-title":"Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway","volume":"91","author":"Nagashima","year":"2017","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1099\/jgv.0.000393","article-title":"Proposed reference sequences for hepatitis E virus subtypes","volume":"97","author":"Smith","year":"2016","journal-title":"J. Gen. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1186\/s13567-017-0483-9","article-title":"Recent knowledge on hepatitis E virus in Suidae reservoirs and transmission routes to human","volume":"48","author":"Pavio","year":"2017","journal-title":"Vet. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.antiviral.2019.01.007","article-title":"Cell culture systems for the study of hepatitis E virus","volume":"163","author":"Meister","year":"2019","journal-title":"Antiviral Res."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Schemmerer, M., Johne, R., Erl, M., Jilg, W., and Wenzel, J.J. (2019). Isolation of Subtype 3c, 3e and 3f-like Hepatitis E Virus Strains Stably Replicating to High Viral Loads in an Optimized Cell Culture System. Viruses, 11.","DOI":"10.3390\/v11060483"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5697","DOI":"10.1128\/JVI.00146-12","article-title":"Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination","volume":"86","author":"Shukla","year":"2012","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1099\/vir.0.007559-0","article-title":"Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells","volume":"90","author":"Yamada","year":"2009","journal-title":"J. Gen. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.jcv.2016.06.010","article-title":"Hepatitis E virus: Assessment of the epidemiological situation in humans in Europe, 2014\/15","volume":"82","author":"Adlhoch","year":"2016","journal-title":"J. Clin. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1111\/jvh.12157","article-title":"An ORF1-rearranged hepatitis E virus derived from a chronically infected patient efficiently replicates in cell culture","volume":"21","author":"Johne","year":"2014","journal-title":"J. Viral Hep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Schemmerer, M., Apelt, S., Trojnar, E., Ulrich, R.G., Wenzel, J.J., and Johne, R. (2016). Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line. Viruses, 8.","DOI":"10.3390\/v8100267"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4225","DOI":"10.1128\/AEM.00951-16","article-title":"Thermal stability of hepatitis E virus estimated by a cell culture method","volume":"82","author":"Johne","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Glitscher, M., Himmelsbach, K., Woytinek, K., Johne, R., Reuter, A., Spiric, J., Schwaben, L., Gr\u00fcnweller, A., and Hildt, E. (2018). Inhibition of Hepatitis E virus spread by the natural compound silvestrol. Viruses, 10.","DOI":"10.3390\/v10060301"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2438","DOI":"10.1073\/pnas.1018878108","article-title":"Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus-host recombinant","volume":"108","author":"Shukla","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"15270","DOI":"10.1073\/pnas.251555098","article-title":"Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element","volume":"98","author":"Emerson","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1552","DOI":"10.1128\/JVI.79.3.1552-1558.2005","article-title":"Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs","volume":"79","author":"Huang","year":"2005","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2585","DOI":"10.1099\/vir.0.81070-0","article-title":"Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV","volume":"86","author":"Huang","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2349","DOI":"10.1073\/pnas.1618424114","article-title":"Entirely plasmid-based reverse genetics system for rotaviruses","volume":"114","author":"Kanai","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1053\/j.gastro.2014.08.040","article-title":"A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients","volume":"147","author":"Debing","year":"2014","journal-title":"Gastroenterology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1073\/pnas.1912307117","article-title":"Robust hepatitis E virus infection and transcriptional response in human hepatocytes","volume":"117","author":"Todt","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1128\/JVI.73.1.251-259.1999","article-title":"Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter","volume":"73","author":"Buchholz","year":"1999","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"i884","DOI":"10.1093\/bioinformatics\/bty560","article-title":"Fastp: an ultra-fast all-in-one FASTQ preprocessor","volume":"34","author":"Chen","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jviromet.2005.07.004","article-title":"A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus","volume":"131","author":"Jothikumar","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1186\/1743-422X-8-487","article-title":"Thermal stability of hepatitis E virus assessed by a molecular biological approach","volume":"8","author":"Schielke","year":"2011","journal-title":"Virol. J."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/9\/3\/157\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:01:36Z","timestamp":1760173296000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/9\/3\/157"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,2,25]]},"references-count":28,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["pathogens9030157"],"URL":"https:\/\/doi.org\/10.3390\/pathogens9030157","relation":{},"ISSN":["2076-0817"],"issn-type":[{"type":"electronic","value":"2076-0817"}],"subject":[],"published":{"date-parts":[[2020,2,25]]}}}