{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T11:46:43Z","timestamp":1792496803855,"version":"4.5.0"},"reference-count":28,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T00:00:00Z","timestamp":1606953600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Hepatitis E virus (HEV) is one of the major etiological agents responsible for acute hepatitis. Hepatitis E virus does not replicate efficiently in mammalian cell cultures, thus a useful model that mimics persistent HEV replication is needed to dissect the molecular mechanism of pathogenesis. Here we report a genotype-3 HEV RNA replicon expressing an EGFP-Zeocin (EZ) resistant gene (p6-EZ) that persistently self-replicated in cell lines of human (Huh-7-S10-3) or hamster (BHK-21) origin after transfection with <jats:italic>in vitro<\/jats:italic> RNA transcripts and subsequent drug screening. Two cell lines, S10-3-EZ and BHK-21-EZ, stably expressed EGFP in the presence of Zeocin during continuous passages. Both genomic and subgenomic HEV RNAs and viral replicase proteins were stably expressed in persistent HEV replicon cells. The values of the cell models in antiviral testing, innate immune RNA sensing and type I IFN in host defense were further demonstrated. We revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host antiviral innate immune sensing during HEV replication. We further demonstrated that treatment with interferon (IFN-\u03b1) or ribavirin significantly reduced expression of replicon RNA in a dose-dependent manner. The availability of the models will greatly facilitate HEV-specific antiviral development, and delineate mechanisms of HEV replication.<\/jats:p>","DOI":"10.3389\/fmicb.2020.603699","type":"journal-article","created":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T08:10:49Z","timestamp":1606983049000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":15,"title":["Stable Expression of a Hepatitis E Virus (HEV) RNA Replicon in Two Mammalian Cell Lines to Assess Mechanism of Innate Immunity and Antiviral Response"],"prefix":"10.3389","volume":"11","author":[{"given":"Ling-Dong","family":"Xu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Peng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen-Ting","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pinglong","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yao-Wei","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2020,12,3]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"478","DOI":"10.2144\/98243pf01","article-title":"Fusion of green fluorescent protein with the Zeocin(TM)-resistance marker allows visual screening and drug selection of transfected eukaryotic cells.","volume":"24","author":"Bennett","year":"1998","journal-title":"Biotechniques"},{"key":"B2","doi-asserted-by":"publisher","first-page":"1025","DOI":"10.1056\/nejmc0903778","article-title":"Persistent carriage of hepatitis E virus in patients with HIV infection.","volume":"361","author":"Dalton","year":"2009","journal-title":"New Engl. J. Med."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1147","DOI":"10.1073\/pnas.1614955114","article-title":"Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles.","volume":"114","author":"Ding","year":"2017","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B4","doi-asserted-by":"crossref","DOI":"10.1128\/mBio.00769-18","article-title":"Identification of the intragenomic promoter controlling hepatitis E Virus subgenomic RNA transcription.","volume":"9","author":"Ding","year":"2018","journal-title":"mBio"},{"key":"B5","doi-asserted-by":"publisher","first-page":"4838","DOI":"10.1128\/jvi.78.9.4838-4846.2004","article-title":"In vitro replication of hepatitis E Virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein.","volume":"78","author":"Emerson","year":"2004","journal-title":"J. Virol."},{"key":"B6","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1002\/rmv.384","article-title":"Hepatitis E virus.","volume":"13","author":"Emerson","year":"2003","journal-title":"Rev. Med. Virol."},{"key":"B7","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1111\/jvh.12156","article-title":"Chronic hepatitis E: a review of the literature.","volume":"21","author":"Fujiwara","year":"2014","journal-title":"J. Viral. Hepat."},{"key":"B8","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1002\/rmv.483","article-title":"Mechanisms of action of ribavirin against distinct viruses.","volume":"16","author":"Graci","year":"2006","journal-title":"Rev. Med. Virol."},{"key":"B9","doi-asserted-by":"publisher","first-page":"3018","DOI":"10.1128\/jvi.02259-06","article-title":"Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo.","volume":"81","author":"Huang","year":"2007","journal-title":"J. Virol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"212","DOI":"10.1016\/j.meegid.2014.06.024","article-title":"Hepeviridae: an expanding family of vertebrate viruses.","volume":"27","author":"Johne","year":"2014","journal-title":"Infect. Genet. Evol."},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008488","article-title":"Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication.","volume":"16","author":"Ju","year":"2020","journal-title":"PLoS Pathog."},{"key":"B12","doi-asserted-by":"publisher","first-page":"3793","DOI":"10.1128\/jvi.03582-14","article-title":"The lysine residues within the human ribosomal protein S17 sequence naturally inserted into the viral nonstructural protein of a unique strain of hepatitis E virus are important for enhanced virus replication.","volume":"89","author":"Kenney","year":"2015","journal-title":"J. Virol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"835","DOI":"10.1086\/655899","article-title":"Characteristics of autochthonous hepatitis e virus infection in solid-organ transplant recipients in France.","volume":"202","author":"Legrand-Abravanel","year":"2010","journal-title":"J. Infect. Dis."},{"key":"B14","doi-asserted-by":"publisher","first-page":"185","DOI":"10.1007\/978-3-642-55541-1_7","article-title":"Swine hepatitis E virus: cross-species infection and risk in xenotransplantation.","volume":"278","author":"Meng","year":"2003","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1005695","article-title":"Expanding host range and cross-species infection of hepatitis E Virus.","volume":"12","author":"Meng","year":"2016","journal-title":"PLoS Pathog."},{"key":"B16","doi-asserted-by":"crossref","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":"B17","doi-asserted-by":"publisher","first-page":"11924","DOI":"10.1128\/jvi.01935-14","article-title":"Hepatitis E virus inhibits Type I interferon induction by ORF1 products.","volume":"88","author":"Nan","year":"2014","journal-title":"J. Virol."},{"key":"B18","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1111\/j.1440-1746.2006.04209.x","article-title":"Prolonged hepatitis E in an immunocompromised patient [3].","volume":"21","author":"P\u00e9ron","year":"2006","journal-title":"J. Gastroenterol. Hepatol."},{"key":"B19","doi-asserted-by":"publisher","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":"B20","doi-asserted-by":"publisher","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. U.S.A."},{"key":"B21","doi-asserted-by":"publisher","first-page":"1112","DOI":"10.1128\/jcm.02002-09","article-title":"Hepatitis E Virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation.","volume":"48","author":"Takahashi","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"B22","doi-asserted-by":"publisher","first-page":"903","DOI":"10.1099\/vir.0.82535-0","article-title":"Development and evaluation of an efficient cell-culture system for Hepatitis E virus.","volume":"88","author":"Tanaka","year":"2007","journal-title":"J. Gen. Virol."},{"key":"B23","doi-asserted-by":"publisher","first-page":"1906","DOI":"10.1128\/jcm.00629-09","article-title":"Development and characterization of a genotype 4 hepatitis E virus cell culture system using a HE-JF5\/15F strain recovered from a fulminant hepatitis patient.","volume":"47","author":"Tanaka","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"2132","DOI":"10.1128\/aac.02427-15","article-title":"Antiviral activities of different interferon types and subtypes against Hepatitis E virus replication.","volume":"60","author":"Todt","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"B25","doi-asserted-by":"publisher","first-page":"1027","DOI":"10.1038\/s41556-019-0352-z","article-title":"HER2 recruits AKT1 to disrupt STING signalling and suppress antiviral defence and antitumour immunity.","volume":"21","author":"Wu","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"4232","DOI":"10.1128\/jvi.02804-15","article-title":"Distinct entry mechanisms for nonenveloped and quasi-enveloped Hepatitis E viruses.","volume":"90","author":"Yin","year":"2016","journal-title":"J. Virol."},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1006417","article-title":"Hepatitis E virus persists in the presence of a type III interferon response.","volume":"13","author":"Yin","year":"2017","journal-title":"PLoS Pathog."},{"key":"B28","doi-asserted-by":"publisher","first-page":"362","DOI":"10.1038\/ncb3496","article-title":"Hippo signalling governs cytosolic nucleic acid sensing through YAP\/TAZ-mediated TBK1 blockade.","volume":"19","author":"Zhang","year":"2017","journal-title":"Nat. Cell Biol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2020.603699\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,12,3]],"date-time":"2020-12-03T08:10:52Z","timestamp":1606983052000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2020.603699\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,12,3]]},"references-count":28,"alternative-id":["10.3389\/fmicb.2020.603699"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2020.603699","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,3]]},"article-number":"603699"}}