scholarly journals Hepatitis E Virus Shows More Genomic Alterations in Cell Culture than In Vivo

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 255 ◽  
Author(s):  
Gulce Sari ◽  
Martijn D.B. van de Garde ◽  
Anne van Schoonhoven ◽  
Jolanda J.C. Voermans ◽  
Annemiek A. van der Eijk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
A549 Cells ◽  
Hepatitis E ◽  
Solid Organ ◽  
Genotype 3 ◽  
Humanized Mouse ◽  
Genomic Alterations

Hepatitis E Virus (HEV) mutations following ribavirin treatment have been associated with treatment non-response and viral persistence, but spontaneous occurring genomic variations have been less well characterized. We here set out to study the HEV genome composition in 2 patient sample types and 2 infection models. Near full HEV genome Sanger sequences of serum- and feces-derived HEV from two chronic HEV genotype 3 (gt3) patients were obtained. In addition, viruses were sequenced after in vitro or in vivo expansion on A549 cells or a humanized mouse model, respectively. We show that HEV acquired 19 nucleotide mutations, of which 7 nonsynonymous amino acids changes located in Open Reading Frame 1 (ORF1), ORF2, and ORF3 coding regions, after prolonged in vitro culture. In vivo passage resulted in selection of 8 nucleotide mutations with 2 altered amino acids in the X domain and Poly-proline region of ORF1. Intra-patient comparison of feces- and serum-derived HEV gt3 of two patients showed 7 and 2 nucleotide mutations with 2 and 0 amino acid changes, respectively. Overall, the number of genomic alterations was up to 1.25× per 1000 nucleotides or amino acids in in vivo samples, and up to 2.84× after in vitro expansion of the same clinical HEV strain. In vitro replication of a clinical HEV strain is therefore associated with more mutations, compared to the minor HEV genomic alterations seen after passage of the same strain in an immune deficient humanized mouse; as well as in feces and blood of 2 immunosuppressed chronically infected HEV patients. These data suggest that HEV infected humanized mice more closely reflect the HEV biology seen in solid organ transplant recipients.

scholarly journals Hepatitis E Virus Immunopathogenesis

Pathogens ◽  
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.

Novel in vivo and in vitro models of Hepatitis E virus genotype 3 infectivity for chronic human HEV infection

2015 ◽  
Vol 70 ◽  
pp. S120
Author(s):  
M.D.B. van de Garde ◽  
S.D. Pas ◽  
G. van der Net ◽  
B.L. Haagmans ◽  
A. Boonstra ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
In Vitro Models ◽  
Genotype 3 ◽  
Virus Genotype

scholarly journals Hepatitis E Virus (HEV) Genotype 3 Infection of Human Liver Chimeric Mice as a Model for Chronic HEV Infection

2016 ◽  
Vol 90 (9) ◽  
pp. 4394-4401 ◽  
Author(s):  
Martijn D. B. van de Garde ◽  
Suzan D. Pas ◽  
Guido van der Net ◽  
Robert A. de Man ◽  
Albert D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Liver ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Chimeric Mouse ◽  
Chimeric Mice ◽  
Genotype 3 ◽  
Edta Plasma

ABSTRACTGenotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities ofin vivoHEV studies in a human liver chimeric mouse model (uPA+/+Nod-SCID-IL2Rγ−/−) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. Thesein vitroHEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 daysin vitro. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV bothin vitroandin vivo. Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans.IMPORTANCEHepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared thein vivoinfectivity of clinical samples from chronic HEV patients in human liver chimeric mice to anin vitrovirus culture system. Efficientin vivoHEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observedin vivoinfectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEV's pathobiology.

scholarly journals Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV

2005 ◽  
Vol 86 (9) ◽  
pp. 2585-2593 ◽  
Author(s):  
F. F. Huang ◽  
F. W. Pierson ◽  
T. E. Toth ◽  
X. J. Meng
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Cdna Clones ◽  
Virus Shedding ◽  
Rna Transcripts ◽  
Successful Infection ◽  
The Usa ◽  
Avian Hev

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis–splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

scholarly journals Ribavirin InhibitsIn VitroHepatitis E Virus Replication through Depletion of Cellular GTP Pools and Is Moderately Synergistic with Alpha Interferon

2013 ◽  
Vol 58 (1) ◽  
pp. 267-273 ◽  
Author(s):  
Yannick Debing ◽  
Suzanne U. Emerson ◽  
Yijin Wang ◽  
Qiuwei Pan ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Hepatitis E Virus ◽  
Clinical Effectiveness ◽  
Hepatitis E ◽  
Alpha Interferon ◽  
Chronic Infections ◽  
Genotype 3 ◽  
Subgenomic Replicon ◽  
First Time

ABSTRACTHepatitis E virus (HEV) is a common cause of acute hepatitis that results in high mortality in pregnant women and may establish chronic infections in immunocompromised patients. We demonstrate for the first time that alpha interferon (IFN-α) and ribavirin inhibitin vitroHEV replication in both a subgenomic replicon and an infectious culture system based on a genotype 3 strain. IFN-α showed a moderate but significant synergism with ribavirin. These findings corroborate the reported clinical effectiveness of both drugs. In addition, the antiviral activity of ribavirin against wild-type genotype 1, 2, and 3 strains was confirmed by immunofluorescence staining. Furthermore, thein vitroactivity of ribavirin depends on depletion of intracellular GTP pools, which is evident from the facts that (i) other GTP-depleting agents (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide [EICAR] and mycophenolic acid) inhibit viral replication, (ii) exogenously added guanosine reverses the antiviral effects, and (iii) a strong correlation (R2= 0.9998) exists between the antiviral activity and GTP depletion of ribavirin and other GTP-depleting agents.

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

Molecular biology and pathogenesis of hepatitis E virus

1999 ◽  
Vol 1 (18) ◽  
pp. 1-16 ◽  
Author(s):  
Shahid Jameel
Keyword(s):  
Viral Genome ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Host Interactions ◽  
Processing Enzymes ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) infection results in hepatitis E, an acute and self-limited disease. The virus is transmitted in a faecal–oral manner and is a major cause of viral hepatitis in much of the developing world, where it causes rampant sporadic infections and large epidemics. A curious feature of hepatitis E is the unusually high rates of mortality that are observed in pregnant women, in whom the disease is exacerbated by the development of fulminant liver disease. In the absence of viable in vitro propagation systems, several geographical isolates of HEV have been maintained in vivo in nonhuman primates and, subsequently, the viral genome has been cloned and sequenced. HEV has been classified provisionally into a separate family known as the HEV-like viruses, which has at least four recognised genotypes, but has only a single serotype. The viral genome is a positive-stranded (+)RNA of ~7.5 kb and encodes at least three proteins. Open reading frame 1 (ORF1) encodes the viral nonstructural polyprotein, which has domains that are homologous to some of the replication and processing enzymes found in other +RNA viruses. The HEV protein itself remains poorly characterised. The protein encoded by open reading frame 2 (ORF2) is the major HEV capsid protein, and the protein encoded by open reading frame 3 (ORF3) appears to be involved in virus–host interactions. Several questions related to the biology, epidemiology and pathogenesis of HEV remain unanswered; the progress of a few of these is reviewed here.

scholarly journals Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells

2009 ◽  
Vol 90 (2) ◽  
pp. 457-462 ◽  
Author(s):  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Yu Hoshino ◽  
Hideyuki Takahashi ◽  
Koji Ichiyama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cdna Clone ◽  
Hepatitis E Virus ◽  
Culture Supernatant ◽  
Cultured Cells ◽  
A549 Cells ◽  
Hepatitis E ◽  
Infectious Cdna Clone ◽  
Post Inoculation ◽  
Genotype 3

A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed in this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 107 copies ml−1 on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 106 copies ml−1 at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.

scholarly journals The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo

2018 ◽  
Vol 157 ◽  
pp. 151-158 ◽  
Author(s):  
Daniel Todt ◽  
Nora Moeller ◽  
Dimas Praditya ◽  
Volker Kinast ◽  
Martina Friesland ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Natural Compound ◽  
Hepatitis E
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