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.

scholarly journals Advances in Hepatitis E Virus Biology and Pathogenesis

Viruses ◽  
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.

scholarly journals Hepatitis E and Pregnancy: An Unholy Alliance Unmasked from Kashmir, India

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1329
Author(s):  
Mohammad Sultan Khuroo
Keyword(s):  
Viral Replication ◽  
Pregnant Women ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Polymerase Activity ◽  
Severe Liver ◽  
Reading Frame ◽  
Multiple Factors ◽  
Severe Liver Disease ◽  
Development And Management

The adverse relationship between viral hepatitis and pregnancy in developing countries had been interpreted as a reflection of retrospectively biased hospital-based data collection by the West. However, the discovery of hepatitis E virus (HEV) as the etiological agent of an epidemic of non-A, non-B hepatitis in Kashmir, and the documenting of the increased incidence and severity of hepatitis E in pregnancy via a house-to-house survey, unmasked this unholy alliance. In the Hepeviridae family, HEV-genotype (gt)1 from genus Orthohepevirus A has a unique open reading frame (ORF)4-encoded protein which enhances viral polymerase activity and viral replication. The epidemics caused by HEV-gt1, but not any other Orthohepevirus A genotype, show an adverse relationship with pregnancy in humans. The pathogenesis of the association is complex and at present not well understood. Possibly multiple factors play a role in causing severe liver disease in the pregnant women including infection and damage to the maternal-fetal interface by HEV-gt1; vertical transmission of HEV to fetus causing severe fetal/neonatal hepatitis; and combined viral and hormone related immune dysfunction of diverse nature in the pregnant women, promoting viral replication. Management is multidisciplinary and needs a close watch for the development and management of acute liver failure. (ALF). Preliminary data suggest beneficial maternal outcomes by early termination of pregnancy in patients with lower grades of encephalopathy.

scholarly journals Association of poor virus specific immunoglobulin G antibody responses with higher viral load is seen in Bangladeshi pregnant women having acute Hepatitis E Genotype 1 infection

2020 ◽  
Author(s):  
Rosy Sultana ◽  
Md Tarikul Islam ◽  
Golam Sarower Bhuyan ◽  
Farjana Akther Noor ◽  
Suprovath Kumar Sarker ◽  
...  
Keyword(s):  
Viral Load ◽  
Acute Hepatitis ◽  
Pregnant Women ◽  
South Asian ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Genotype 1 ◽  
Asian Countries ◽  
Igg Antibody ◽  
Acute Hepatitis E

AbstractAlthough Hepatitis E viral illness is usually self-limiting, higher rates of morbidity and mortality are frequently observed during pregnancy in South Asian countries including Bangladesh. Of the four common variants, hepatitis E virus genotype 1 is mainly prevalent in South Asian countries. Pregnant women usually suffer from a state of immunosuppression. It is yet to be known whether virus specific immunoglobulin G (IgG) immune responses have any association with the vulnerability of pregnant women to acute hepatitis with E virus. The study aimed to compare the viral load and IgG responses of hepatitis E-infected pregnant women with that of non-pregnant women with same infection. Real Time –quantitative reverse transcription Polymerase Chain Reaction and Sanger sequencing were performed to determine the viral load and genotype, respectively, whereas Enzyme Linked Immunosorbent Assay method was used to determine hepatitis E virus specific serum IgG antibody index along with IgG avidity index. Although significant negative correlations were observed between log viral copy number and log IgG antibody index in the late acute phases of jaundice for both pregnant (r= −0.7971, p=0.0002) and non-pregnant women (r= −0.9117, p=0.0002), serum log viral copy number of pregnant women was significantly higher than that of the non-pregnant counterpart (p=0.0196) in the late acute stage of jaundice. In addition, log hepatitis E virus IgG antibody index of pregnant women was significantly lower than the non-pregnant women in the late phase of jaundice induced by hepatitis E virus (p=0.0303). Moreover, pregnant women with acute hepatitis E had higher cross-reactive IgG than in the non-pregnant women (p=0.0017). All the patients got infected with hepatitis E virus were in Genotype 1 variety. The study demonstrates that virus-specific poor IgG responses might be responsible for vulnerability of pregnant women to acute hepatitis with hepatitis E virus.Author SummaryAcute hepatitis caused by hepatitis E virus (HEV) Genotype 1 is a public health problem in Asian countries and especially it poses a potential health threat to pregnant women causing 19% to 25% mortality, particularly in South Asian countries including Bangladesh. The study aimed to explore whether HEV IgG immune responses were compromised during pregnancy, which might contribute to higher viral load and disease severity. Accordingly, pregnant and non-pregnant women with acute hepatitis (clinically presented with nausea, loss of appetite and /or jaundice) were enrolled from different tertiary care hospitals in Dhaka city. All these patients were screened and hepatitis E were differentiated from other hepatitis (caused by A, B, C) using Enzyme Linked Immunosorbent Assay (ELISA) methods. HEV IgG antibody/avidity indices and viral loads were measured using ELISA and real time quantitative polymerase chain reaction (RT-qPCR), respectively. The study showed that pregnant women with acute hepatitis E had lower IgG indices with higher viral load than their non-pregnant counterpart. Overall, the study revealed that virus-specific poor IgG responses might render pregnant women vulnerable to acute hepatitis E of varying degree of severity which might be associated with higher viral load.

Hepatitis E virus genotyping in Pakistan: a regional study to explore the implications for pregnant females

2021 ◽  
Author(s):  
Mahnoor Nadeem ◽  
Tahir Ahmad ◽  
Salik Javed Kakar ◽  
Fazal Adnan ◽  
Sadia Anjum
Keyword(s):  
Pregnant Women ◽  
Hepatitis E Virus ◽  
Acute Infection ◽  
Hepatitis E ◽  
Immunoglobulin M ◽  
Genotype 1 ◽  
Regional Study ◽  
Third Trimester ◽  
Pregnant Females ◽  
Diagnostic Center

Aim: Hepatitis E virus (HEV) has different genotypes 1–4, which is generally associated with mild to severe complications among immunocompromised patients and pregnant women. Materials & methods: Immunoglobulin M (IgM) HEV-positive samples were collected from the diagnostic center. HEV infection was further confirmed by RT-based PCR and genotyping was done to affirm the prevailing genotype. Results: This study identified 28 patients from Islamabad who were confirmed to have immunoglobulins type M against HEV showing acute infection, of which 17 were pregnant and 11 were non-pregnant women. All pregnant women were in their third trimester of pregnancy. Conclusion: Genotype-1 is commonly associated with pregnant females presenting with HEV infections in Islamabad. There is a need to further identify both the sources & route of infections.

Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication

2018 ◽  
Vol 25 (6) ◽  
pp. 742-751 ◽  
Author(s):  
C. Yang ◽  
W. Yu ◽  
Y. Bi ◽  
F. Long ◽  
Y. Li ◽  
...  
Keyword(s):  
Viral Replication ◽  
Pregnant Women ◽  
Hepatitis E Virus ◽  
Hepatitis E

scholarly journals Antiviral Candidates for Treating Hepatitis E Virus Infection

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Natalie E. Netzler ◽  
Daniel Enosi Tuipulotu ◽  
Subhash G. Vasudevan ◽  
Jason M. Mackenzie ◽  
Peter A. White
Keyword(s):  
Hepatitis E Virus ◽  
Drug Repurposing ◽  
Hepatitis E ◽  
Genotype 1 ◽  
Rna Quantification ◽  
Half Maximal Effective Concentration ◽  
Combinational Treatment ◽  
Further Development ◽  
Rna Levels

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.

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

2013 ◽  
Vol 88 (2) ◽  
pp. 868-877 ◽  
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

scholarly journals Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 406
Author(s):  
Marie Pellerin ◽  
Edouard Hirchaud ◽  
Yannick Blanchard ◽  
Nicole Pavio ◽  
Virginie Doceul
Keyword(s):  
Cell Culture ◽  
Culture System ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Inhibitory Effect ◽  
Cell Culture System ◽  
Efficient Replication ◽  
A Cell ◽  
Vitro Model

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.

scholarly journals Proteomic Comparative Analysis of Pregnancy Serum Facilitating Hepatitis E Virus Replication in Hepatoma Cells

2017 ◽  
Author(s):  
Yi Li ◽  
Yanhong Bi ◽  
Wenhai Yu ◽  
Chenchen Yang ◽  
Jue Wang ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Pregnant Women ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Hepatoma Cells ◽  
Host Cells ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Absolute Quantitation

Hepatitis E virus (HEV) is a common cause of acute hepatitis worldwide, accounting for approximately 25% of deaths among pregnant women. We previously reported that pregnancy serum facilitates HEV replication in vitro. However, the differences in host cells with HEV infection induced by pregnancy serum and fetal bovine serum (FBS) are unclear. In this study, differentially expressed proteins were identified in HEV-infected hepatoma cells (HepG2) supplemented with different sera by using isobaric tags for relative and absolute quantitation. Proteomic analysis indicated that HEV infection significantly induced 1014 differentially expressed proteins in HEV-infected HepG2 cells when supplemented with FBS compared with pregnancy serum. Further validation by Western blot confirmed that filamin A, heat-shock proteins 70 and 90, Cytochrome c, and Thioredoxin were associated with HEV infection. This comparative analysis provides an important basis to further investigate HEV pathogenesis in pregnant women and HEV replication.

scholarly journals Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

2017 ◽  
pp. JVI.01932-17 ◽  
Author(s):  
Shubhra Agarwal ◽  
Prasith Baccam ◽  
Rakesh Aggarwal ◽  
Naga Suresh Veerapu
Keyword(s):  
Genetic Diversity ◽  
Cell Culture ◽  
Hepatitis E Virus ◽  
Rna Viruses ◽  
Hepatitis E ◽  
Replication Capacity ◽  
Genotype 1 ◽  
Wild Type ◽  
Virus Genotype ◽  
Cross Sectional

Many RNA viruses exist as an ensemble of genetically-diverse, replicating populations, known as mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds of varying genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 (ORF1) or of the full-genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency of the order of 10-3per nucleotide copied, and existence of marked genetic diversity with a high normalized Shannon entropy. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of varying sizes and compositions on phenotypic characteristics such as the replication level of (+)-RNA progeny, amount of double-stranded RNA (a surrogate for rate of viral replication) and ORF1 protein, and expression of interferon stimulating genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help provide further insights into virus evolution and prediction of emerging viral diseases.IMPORTANCESeveral biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloudin vitro. We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a ss(+)RNA virus, with varying and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effect of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud, which had an extremely highgenetic diversity, had replication rate exceeding that of the wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

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