Antiviral Candidates for Treating Hepatitis E Virus Infection

ABSTRACT Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC50], 0.03 μM; half-maximal cytotoxic concentration [CC50], >100 μM) and GPC-N114 (EC50, 1.07 μM, CC50, >100 μM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 μM GPC-N114 or 2.50 μM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC50, 1.97 μM; CC50, >100 μM) and reduced replicon RNA levels (47.2% reduction at 10 μM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.

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

Viruses ◽

10.3390/v13010075 ◽

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.

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Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

Journal of Virology ◽

10.1128/jvi.02205-13 ◽

2013 ◽

Vol 88 (2) ◽

pp. 868-877 ◽

Cited By ~ 25

Author(s):

H. T. Nguyen ◽

P. Shukla ◽

U. Torian ◽

K. Faulk ◽

S. U. Emerson

Keyword(s):

Hepatitis E Virus ◽

Hepatitis E ◽

Kidney Cells ◽

Genotype 1 ◽

Virus Genotype ◽

Multiple Levels ◽

Swine Kidney

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In Vitro and In Vivo Mutational Analysis of the 3′-Terminal Regions of Hepatitis E Virus Genomes and Replicons

Journal of Virology ◽

10.1128/jvi.79.2.1017-1026.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 1017-1026 ◽

Cited By ~ 47

Author(s):

Judith Graff ◽

Hanh Nguyen ◽

Chaiyan Kasorndorkbua ◽

Patrick G. Halbur ◽

Marisa St. Claire ◽

...

Keyword(s):

Hepatitis E Virus ◽

Cultured Cells ◽

Mutational Analysis ◽

Rna Replication ◽

Hepatitis E ◽

Human Strain ◽

Full Length ◽

Genotype 1

ABSTRACT Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3′-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3′-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3′-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3′ termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3′ terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3′-terminal mutations in vivo. Incorporation of the 3′-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

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Complete Genome Sequence of a Hepatitis E Virus Genotype 1e Strain from an Outbreak in Nigeria, 2017

Microbiology Resource Announcements ◽

10.1128/mra.01378-18 ◽

2019 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Olusola Anuoluwapo Akanbi ◽

Dominik Harms ◽

Bo Wang ◽

Oluyinka Oladele Opaleye ◽

Olufisayo Adesina ◽

...

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Sub Saharan Africa ◽

Genotype 1 ◽

Virus Genotype ◽

Sub Saharan

Hepatitis E virus genotype 1 (HEV-1) is associated with large epidemics. Notably, HEV subtype 1e (HEV-1e) has caused HEV outbreaks in sub-Saharan Africa.

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Advances in Hepatitis E Virus Biology and Pathogenesis

Viruses ◽

10.3390/v13020267 ◽

2021 ◽

Vol 13 (2) ◽

pp. 267

Author(s):

Shaoli Lin ◽

Yan-Jin Zhang

Keyword(s):

Pregnant Women ◽

Hepatitis E Virus ◽

Case Fatality ◽

Hepatitis E ◽

Host Cells ◽

High Rate ◽

Rapid Progression ◽

Genotype 1 ◽

Genotype 3 ◽

Zoonotic Infections

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.

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Evaluation of hepatitis E antigen kinetics and its diagnostic utility for prediction of the outcomes of hepatitis E virus genotype 1 infection

Virulence ◽

10.1080/21505594.2021.1922027 ◽

2021 ◽

Vol 12 (1) ◽

pp. 1334-1344

Author(s):

Mohamed A. El-Mokhtar ◽

Haidi Karam-Allah Ramadan ◽

Muhamad R. Abdel Hameed ◽

Ayat M. Kamel ◽

Sahar A. Mandour ◽

...

Keyword(s):

Hepatitis E Virus ◽

Hepatitis E ◽

Diagnostic Utility ◽

Genotype 1 ◽

Virus Genotype

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1354 IN VITRO INFECTION AND REPLICATION OF HEPATITIS E VIRUS IN HUMAN HEPATOCYTES

Journal of Hepatology ◽

10.1016/s0168-8278(11)61356-1 ◽

2011 ◽

Vol 54 ◽

pp. S535

Author(s):

Y. Oshiro ◽

H. Yasue ◽

S. Hattori ◽

M. Chiba ◽

T. Naito ◽

...

Keyword(s):

Hepatitis E Virus ◽

Hepatitis E ◽

Human Hepatocytes

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Hepatitis E Virus Immunopathogenesis

Pathogens ◽

10.3390/pathogens10091180 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1180

Author(s):

Kush Kumar Yadav ◽

Scott P. Kenney

Keyword(s):

Hepatitis E Virus ◽

Organ Transplant ◽

Hepatitis E ◽

Oral Route ◽

Solid Organ ◽

Immunocompromised Patients ◽

In Vivo Models ◽

Transplant Patients

Hepatitis E virus is an important emerging pathogen producing a lethal impact on the pregnant population and immunocompromised patients. Starting in 1983, it has been described as the cause for acute hepatitis transmitted via the fecal–oral route. However, zoonotic and blood transfusion transmission of HEV have been reported in the past few decades, leading to the detailed research of HEV pathogenesis. The reason behind HEV being highly virulent to the pregnant population particularly during the third trimester, leading to maternal and fetal death, remains unknown. Various host factors (immunological, nutritional, hormonal) and viral factors have been studied to define the key determinants assisting HEV to be virulent in pregnant and immunocompromised patients. Similarly, chronic hepatitis is seen particularly in solid organ transplant patients, resulting in fatal conditions. This review describes recent advances in the immunopathophysiology of HEV infections in general, pregnant, and immunocompromised populations, and further elucidates the in vitro and in vivo models utilized to understand HEV pathogenesis.

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DESIGN OF HEPATITIS E VIRUS GENOTYPE 1 RECOMBINANT ORF3 PROTEIN BY CODON OPTIMIZATION METHOD

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2017-6-63-72 ◽

2017 ◽

pp. 63-72

Author(s):

G. I. Alatortseva ◽

A. V. Sidorov ◽

L. N. Nesterenko ◽

L. N. Luhverchik ◽

M. V. Zhukina ◽

...

Keyword(s):

Recombinant Protein ◽

Hepatitis E Virus ◽

Optimization Method ◽

Hepatitis E ◽

Antigenic Specificity ◽

Clinical Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Genotype 1 ◽

Recombinant Polypeptide ◽

Full Size

Aim. The development of the hepatitis E virus (HEV) genotype 1 full-size ORF3 recombinant polypeptide. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. HEV genotype 1 RNA had been isolated from clinical samples collected in Kyrgyzstan. DNA copy of subgenomic virus RNA had been cloned and used for further development of E.coli strains producing full-size recombinant protein ORF3 fused to E.coli beta-galactosidase. Codons optimization method was used in aim to increase expression level of recombinant protein. Recombinant protein ORF3 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. Antigenic specificity of recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay and Western blotting with the specific sera. Conclusion. HEVgenotype 1 ORF3 recombinant antigen had been designed, and it’s applicability in diagnostic tests had been experimentally confirmed.

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