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Optimized Hepatitis E Virus (HEV) Culture and its Application to Measurements of HEV Infectivity.

ABSTRACT: Hepatitis E virus (HEV) is a major concern in public health worldwide. Infections with HEV genotypes 3, 4, or 7 can lead to chronic hepatitis while genotype 1 infections can trigger severe hepatitis in pregnant women. Infections with all genotypes can worsen chronic liver diseases. As virions are lipid-associated in blood and naked in feces, efficient methods of propagating HEV clinical strains in vitro and evaluating the infectivity of both HEV forms are needed. We evaluated the spread of clinical strains of HEV genotypes 1 (HEV1) and 3 (HEV3) by quantifying viral RNA in culture supernatants and cell lysates. Infectivity was determined by endpoint dilution and calculation of the tissue culture infectious dose 50 (TCID50). An enhanced HEV production could be obtained varying the composition of the medium, including fetal bovine serum (FBS) and dimethylsulfoxide (DMSO) content. This increased TCID50 from 10 to 100-fold and allowed us to quantify HEV1 infectivity. These optimized methods for propagating and measuring HEV infectivity could be applied to health safety processes and will be useful for testing new antiviral drugs.

SUBMITTER: Capelli N

PROVIDER: S-EPMC7077187 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Optimized Hepatitis E Virus (HEV) Culture and its Application to Measurements of HEV Infectivity.

Capelli Nicolas N Dubois Martine M Pucelle Mélanie M Da Silva Isabelle I Lhomme Sébastien S Abravanel Florence F Chapuy-Regaud Sabine S Izopet Jacques J

Viruses 20200124 2

Hepatitis E virus (HEV) is a major concern in public health worldwide. Infections with HEV genotypes 3, 4, or 7 can lead to chronic hepatitis while genotype 1 infections can trigger severe hepatitis in pregnant women. Infections with all genotypes can worsen chronic liver diseases. As virions are lipid-associated in blood and naked in feces, efficient methods of propagating HEV clinical strains in vitro and evaluating the infectivity of both HEV forms are needed. We evaluated the spread of clini ...[more]

PMID: 31991673

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Project description: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.

Dataset Information

Optimized Hepatitis E Virus (HEV) Culture and its Application to Measurements of HEV Infectivity.

Publications

Optimized Hepatitis E Virus (HEV) Culture and its Application to Measurements of HEV Infectivity.

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