{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T17:45:55Z","timestamp":1792691155981,"version":"4.5.0"},"reference-count":37,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,4,26]],"date-time":"2021-04-26T00:00:00Z","timestamp":1619395200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008751","name":"Bundesinstitut f\u00fcr Risikobewertung","doi-asserted-by":"publisher","award":["1322-686"],"award-info":[{"award-number":["1322-686"]}],"id":[{"id":"10.13039\/501100008751","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003107","name":"Bundesministerium f\u00fcr Gesundheit","doi-asserted-by":"publisher","award":["ZMVI1-2518FSB705"],"award-info":[{"award-number":["ZMVI1-2518FSB705"]}],"id":[{"id":"10.13039\/501100003107","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.<\/jats:p>","DOI":"10.3390\/v13050762","type":"journal-article","created":{"date-parts":[[2021,4,27]],"date-time":"2021-04-27T06:19:11Z","timestamp":1619504351000},"page":"762","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["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"],"prefix":"10.3390","volume":"13","author":[{"given":"Johannes","family":"Scholz","sequence":"first","affiliation":[{"name":"German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Falkenhagen","sequence":"additional","affiliation":[{"name":"German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9597-6724","authenticated-orcid":false,"given":"Reimar","family":"Johne","sequence":"additional","affiliation":[{"name":"German Federal Institute for Risk Assessment, Max-Dohrn-Stra\u00dfe 8-10, 10589 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2477","DOI":"10.1016\/S0140-6736(11)61849-7","article-title":"Hepatitis E","volume":"379","author":"Kamar","year":"2012","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"59","DOI":"10.2147\/IDR.S289766","article-title":"Clinical Outcomes and Prevalence of Hepatitis E Virus (HEV) Among Non-A-C Hepatitis Patients in Egypt","volume":"14","author":"Sayed","year":"2021","journal-title":"Infect. Drug Resist."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Khuroo, M.S., Khuroo, M.S., and Khuroo, N.S. (2016). Transmission of Hepatitis E Virus in Developing Countries. Viruses, 8.","DOI":"10.3390\/v8090253"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1002\/rmv.482","article-title":"Phylogenetic analysis of global hepatitis E virus sequences: Genetic diversity, subtypes and zoonosis","volume":"16","author":"Lu","year":"2006","journal-title":"Rev. Med. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5543","DOI":"10.3748\/wjg.v26.i37.5543","article-title":"Hepatitis E virus: Epidemiology, diagnosis, clinical manifestations, and treatment","volume":"26","author":"Aslan","year":"2020","journal-title":"World J. Gastroenterol."},{"key":"ref_6","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_7","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1099\/jgv.0.001435","article-title":"Update: Proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A)","volume":"101","author":"Smith","year":"2020","journal-title":"J. Gen. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Pallerla, S.R., Harms, D., Johne, R., Todt, D., Steinmann, E., Schemmerer, M., Wenzel, J.J., Hofmann, J., Shih, J.W.K., and Wedemeyer, H. (2020). Hepatitis E Virus Infection: Circulation, Molecular Epidemiology, and Impact on Global Health. Pathogens, 9.","DOI":"10.3390\/pathogens9100856"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Oechslin, N., Moradpour, D., and Gouttenoire, J. (2020). On the Host Side of the Hepatitis E Virus Life Cycle. Cells, 9.","DOI":"10.3390\/cells9051294"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5919","DOI":"10.1128\/JVI.00046-06","article-title":"A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus","volume":"80","author":"Graff","year":"2006","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/0042-6822(91)90760-9","article-title":"Hepatitis E virus (HEV): Molecular cloning and sequencing of the full-length viral genome","volume":"185","author":"Tam","year":"1991","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Gouttenoire, J., Pollan, A., Abrami, L., Oechslin, N., Mauron, J., Matter, M., Oppliger, J., Szkolnicka, D., Dao Thi, V.L., and van der Goot, F.G. (2018). Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion. PLoS Pathog., 14.","DOI":"10.1371\/journal.ppat.1007471"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1128\/jvi.70.1.207-216.1996","article-title":"Expression in animal cells and characterization of the hepatitis E virus structural proteins","volume":"70","author":"Jameel","year":"1996","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8259","DOI":"10.1073\/pnas.89.17.8259","article-title":"Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: Delineation of an additional group of positive-strand RNA plant and animal viruses","volume":"89","author":"Koonin","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10186","DOI":"10.1128\/JVI.01374-12","article-title":"Evolution of the hepatitis E virus polyproline region: Order from disorder","volume":"86","author":"Purdy","year":"2012","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Purdy, M.A., Lara, J., and Khudyakov, Y.E. (2012). The hepatitis E virus polyproline region is involved in viral adaptation. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0035974"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"10031","DOI":"10.1128\/JVI.00763-11","article-title":"Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication","volume":"85","author":"Pudupakam","year":"2011","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Munoz-Chimeno, M., Cenalmor, A., Garcia-Lugo, M.A., Hernandez, M., Rodriguez-Lazaro, D., and Avellon, A. (2020). Proline-Rich Hypervariable Region of Hepatitis E Virus: Arranging the Disorder. Microorganisms, 8.","DOI":"10.3390\/microorganisms8091417"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fmicb.2020.00001","article-title":"Insertions and Duplications in the Polyproline Region of the Hepatitis E Virus","volume":"11","author":"Lhomme","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_20","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_21","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1099\/vir.0.037259-0","article-title":"A naturally occurring human\/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture","volume":"93","author":"Nguyen","year":"2012","journal-title":"J. Gen. Virol."},{"key":"ref_22","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_23","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 Hepat."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4225","DOI":"10.1128\/AEM.00951-16","article-title":"Thermal Stability of Hepatitis E Virus as Estimated by a Cell Culture Method","volume":"82","author":"Johne","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"109013","DOI":"10.1016\/j.ijfoodmicro.2020.109013","article-title":"Stability of hepatitis E virus at high hydrostatic pressure processing","volume":"339","author":"Johne","year":"2021","journal-title":"Int. J. Food Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1007\/s12560-020-09440-2","article-title":"Effect of Sodium Chloride, Sodium Nitrite and Sodium Nitrate on the Infectivity of Hepatitis E Virus","volume":"12","author":"Wolff","year":"2020","journal-title":"Food Environ. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"108625","DOI":"10.1016\/j.ijfoodmicro.2020.108625","article-title":"Stability of hepatitis E virus at different pH values","volume":"325","author":"Wolff","year":"2020","journal-title":"Int. J. Food Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Glitscher, M., Himmelsbach, K., Woytinek, K., Johne, R., Reuter, A., Spiric, J., Schwaben, L., Grunweller, A., and Hildt, E. (2018). Inhibition of Hepatitis E Virus Spread by the Natural Compound Silvestrol. Viruses, 10.","DOI":"10.3390\/v10060301"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Glitscher, M., Himmelsbach, K., Woytinek, K., Schollmeier, A., Johne, R., Praefcke, G.J.K., and Hildt, E. (2021). Identification of the interferon-inducible GTPase GBP1 as major restriction factor for the Hepatitis E virus. J. Virol.","DOI":"10.1128\/JVI.01564-20"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e15","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_31","doi-asserted-by":"crossref","first-page":"1733","DOI":"10.1136\/gutjnl-2015-311000","article-title":"In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome","volume":"65","author":"Todt","year":"2016","journal-title":"Gut"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Scholz, J., Bachlein, C., Gadicherla, A.K., Falkenhagen, A., Tausch, S.H., and Johne, R. (2020). Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c. Pathogens, 9.","DOI":"10.3390\/pathogens9030157"},{"key":"ref_33","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_34","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_35","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_36","doi-asserted-by":"crossref","first-page":"1","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."},{"key":"ref_37","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"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/5\/762\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:53:06Z","timestamp":1760161986000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/5\/762"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,26]]},"references-count":37,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["v13050762"],"URL":"https:\/\/doi.org\/10.3390\/v13050762","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,26]]}}}