P200: Extrahepatic replication of hepatitis E virus in neuronal and placental cell lines

2015 ◽  
Vol 22 ◽  
pp. 120-121
Keyword(s):  
Cell Lines ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Placental Cell ◽  
Extrahepatic Replication

scholarly journals Stable Expression of a Hepatitis E Virus (HEV) RNA Replicon in Two Mammalian Cell Lines to Assess Mechanism of Innate Immunity and Antiviral Response

2020 ◽  
Vol 11 ◽  
Author(s):  
Ling-Dong Xu ◽  
Fei Zhang ◽  
Lei Peng ◽  
Wen-Ting Luo ◽  
Chu Chen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Innate Immune ◽  
Type I ◽  
Dependent Manner ◽  
Genotype 3 ◽  
Mammalian Cell Lines ◽  
Rna Replicon

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 in vitro 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-α) 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.

scholarly journals Evidence of the Extrahepatic Replication of Hepatitis E Virus in Human Endometrial Stromal Cells

Pathogens ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 295 ◽  
Author(s):  
Mohamed A. El-Mokhtar ◽  
Essam R. Othman ◽  
Maha Y. Khashbah ◽  
Ali Ismael ◽  
Mohamed AA Ghaliony ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Stromal Cells ◽  
Hepatitis E Virus ◽  
Inflammatory Responses ◽  
Hepatitis E ◽  
Endometrial Stromal Cells ◽  
Extrahepatic Replication ◽  
Efficient Replication ◽  
Over Time

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The tropism of HEV is not restricted to the liver, and the virus replicates in other organs. Not all the extrahepatic targets for HEV are identified. Herein, we found that non-decidualized primary human endometrial stromal cells (PHESCs), which are precursors for the decidua and placenta, are susceptible to HEV infection. PHESCs, isolated from healthy non-pregnant women (n = 5), were challenged with stool-derived HEV-1 and HEV-3. HEV RNA was measured by qPCR, and HEV capsid protein was assessed by flow cytometry, immunofluorescence (IF), and ELISA. HEV infection was successfully established in PHESCs. Intracellular and extracellular HEV RNA loads were increased over time, indicating efficient replication in vitro. In addition, HEV capsid protein was detected intracellularly in the HEV-infected PHESCs and accumulated extracellularly over time, confirming the viral assembly and release from the infected cells. HEV-1 replicated more efficiently in PHESCs than HEV-3 and induced more inflammatory responses. Ribavirin (RBV) treatment abolished the replication of HEV in PHESCs. In conclusion, PHESCs are permissive to HEV infection and these cells could be an endogenous source of HEV infection during pregnancy and mediate HEV vertical transmission.

scholarly journals Evidence of extrahepatic replication of hepatitis E virus in human placenta

2014 ◽  
Vol 95 (6) ◽  
pp. 1266-1271 ◽  
Author(s):  
Purabi Deka Bose ◽  
Bhudev Chandra Das ◽  
Rajib Kishore Hazam ◽  
Ashok Kumar ◽  
Subhash Medhi ◽  
...  
Keyword(s):  
Human Placenta ◽  
Hepatitis E Virus ◽  
Placental Tissue ◽  
Hepatitis E ◽  
Fetal Mortality ◽  
Positive Staining ◽  
Structural Protein ◽  
Tissue Sections ◽  
Extrahepatic Replication ◽  
Immunohistochemical Studies

The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with high fetal mortality. Based on this evidence and in the absence of reports on HEV replication in extrahepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative-strand-specific reverse transcriptase PCR. Viral load was estimated by real-time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in blood samples. Positive staining of placental tissue sections with HEV antibody against the viral structural protein ORF3 was observed. HEV replication in placenta also correlated with fetal and maternal mortality in ALF patients. It is demonstrated for the first time that HEV replication occurs in human placenta and that placenta may be a site of extrahepatic replication of HEV in humans.

Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines

2014 ◽  
Vol 185 ◽  
pp. 92-102 ◽  
Author(s):  
Suljid Jirintai ◽  
Tanggis ◽  
Mulyanto ◽  
Joseph Benedictus Suparyatmo ◽  
Masaharu Takahashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hepatitis E Virus ◽  
Hepatoma Cell ◽  
Rattus Rattus ◽  
Hepatitis E ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Hepatoma Cell Lines ◽  
Wild Rats

scholarly journals Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 75
Author(s):  
Kush K. Yadav ◽  
Patricia A. Boley ◽  
Zachary Fritts ◽  
Scott P. Kenney
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Pregnant Women ◽  
Cell Lines ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Target Cells ◽  
Genotype 1 ◽  
Orf4 Protein

Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

P686 EXTRAHEPATIC REPLICATION OF HEPATITIS E VIRUS IN HUMAN PLACENTA: ITS CLINICAL IMPLICATIONS

2014 ◽  
Vol 60 (1) ◽  
pp. S297-S298
Author(s):  
P. Deka ◽  
B.C. Das ◽  
R.K. Hazam ◽  
A. Kumar ◽  
P. Kar
Keyword(s):  
Human Placenta ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Clinical Implications ◽  
Extrahepatic Replication

scholarly journals New models of hepatitis E virus replication in human and porcine hepatocyte cell lines

2013 ◽  
Vol 94 (3) ◽  
pp. 549-558 ◽  
Author(s):  
Sophie Rogée ◽  
Neil Talbot ◽  
Thomas Caperna ◽  
Jérôme Bouquet ◽  
Elodie Barnaud ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hepatitis E Virus ◽  
Embryonic Stem ◽  
Hepatitis E ◽  
Species Barrier ◽  
Barrier Crossing ◽  
New Models ◽  
Culture Models

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

scholarly journals Putative receptor-binding sites of hepatitis E virus

2008 ◽  
Vol 89 (1) ◽  
pp. 245-249 ◽  
Author(s):  
Shuizhen He ◽  
Ji Miao ◽  
Zizheng Zheng ◽  
Ting Wu ◽  
Minghui Xie ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Recombinant Protein ◽  
Cell Lines ◽  
Receptor Binding ◽  
Binding Sites ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Structural Protein ◽  
Mutant Peptide ◽  
Putative Receptor

A truncated structural protein of hepatitis E virus (HEV), p239, occurs as 23 nm particles consisting of partial homodimers. As the latter resemble the HEV capsomere structurally and antigenically, it was postulated that the recombinant protein may serve as a probe for the HEV receptor. This hypothesis was supported by findings that purified p239 bound and penetrated different cell lines that are susceptible to HEV, and inhibited HEV infection of these cells. The binding was blocked by four of six monoclonal antibodies (mAbs) reactive against the dimeric domain of p239, and by two of three mAbs reactive against its monomeric domain, suggesting that binding may involve a portion of each domain. Mutation affecting the monomeric domain had no effect on binding or capacity to block HEV infection, whereas that affecting the dimeric domain diminished binding of the mutant peptide markedly and abrogated its capacity to block HEV infection. These results suggest that HEV infection might involve distinct receptor-binding sites.

scholarly journals A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E Virus

2006 ◽  
Vol 80 (12) ◽  
pp. 5919-5926 ◽  
Author(s):  
Judith Graff ◽  
Udana Torian ◽  
Hanh Nguyen ◽  
Suzanne U. Emerson
Keyword(s):  
Resistance Gene ◽  
Cell Lines ◽  
Northern Blot ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frames ◽  
Subgenomic Rna ◽  
Neomycin Resistance Gene ◽  
Neomycin Resistance ◽  
Reading Frames

ABSTRACT Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

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