scholarly journals DESIGN OF HEPATITIS E VIRUS GENOTYPE 1 RECOMBINANT CAPSID PROTEIN: CLONING, EXPRESSION, PURIFICATION, EVALUATION OF THE ANTIGENIC PROPERTIES

2017 ◽  
pp. 72-80 ◽  
Author(s):  
G. I. Alatortseva ◽  
A. V. Sidorov ◽  
L. N. Nesterenko ◽  
L. N. Luhverchik ◽  
V. V. Dotsenko ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Size Exclusion ◽  
Antigenic Specificity ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Genotype 1 ◽  
Recombinant Polypeptide ◽  
Antigenic Properties

Aim. The development of the hepatitis E virus (HEV) genotype 1 recombinant capsid protein. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. Using HEV genotype 1 DNA copy of subgenomic virus RNA we made E.coli strains producing recombinabt capsid protein, containing C-terminal fragment of ORF2 protein fused to E.coli beta-galactosidase. Recombinant protein ORF2 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. By Western blotting it had been shown specific interaction of the recombinant polypeptide with anti-HEV IgG from pool of positive sera. Antigenic specificity of the recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay with sera of hepatitis E patients and reference groups: healthy donors, patients with hepatitis А, В, C, infectious mononucleosis and cytomegalovirus infection, HIV-infected patients. Conclusion. HEV genotype 1 ORF2 recombinant antigen had been developed, and its possible use in diagnostic tests had been experimentally shown.

scholarly journals DEVELOPMENT OF HEPATITIS E 3 GENOTYPE RECOMBINANT PROTEIN CAPSID OF: CLONING, EXPRESSION, PURIFICATION, EVALUATION OF THE ANTIGENIC PROPERTIES

2019 ◽  
Vol 1 (1) ◽  
pp. 10-17 ◽  
Author(s):  
G. I. Alatortseva ◽  
A. V. Sidorov ◽  
L. N. Nesterenko ◽  
L. N. Luhverchik ◽  
V. V. Dotsenko ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Capsid Protein ◽  
Hepatitis E ◽  
Size Exclusion ◽  
Antigenic Specificity ◽  
Clinical Samples ◽  
Genotype 3 ◽  
Recombinant Polypeptide ◽  
Antigenic Properties ◽  
Exclusion Chromatography

Aim. The development of the hepatitis E virus (HEV) genotype 3 recombinant capsid protein.Materials and methods. E.coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods.Results. Using viruscontaining material from pigs of Belgorod region (Russian Federation) we made E.coli strains producing recombinant capsid protein, containing C-terminal of viral ORF2 protein fragment fused to E.coli β-galactosidase. Recombinant protein ORF2 had been isolated from the bacterial inclusion bodies and purified by size exclusion chromatography. Antigenic specificity of the recombinant polypeptide was confirmed by ELISA and Western blotting with sera of hepatitis E patients and reference groups (healthy donors, patients with hepatitis A, B, C, infectious mononucleosis, cytomegalovirus infection and HIV-infected patients). Conclusion. HEV genotype 3 ORF2 recombinant antigen had been developed, and the possibility to use it in diagnostic tests had been experimentally shown.

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

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.

scholarly journals Detection of Hepatitis E Virus-Specific Immunoglobulin A in Patients Infected with Hepatitis E Virus Genotype 1 or 3

2007 ◽  
Vol 14 (3) ◽  
pp. 276-280 ◽  
Author(s):  
M. Herremans ◽  
E. Duizer ◽  
E. Jusic ◽  
M. P. G. Koopmans
Keyword(s):  
Acute Hepatitis ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Genotype 1 ◽  
Genotype 3 ◽  
Route Of Transmission

ABSTRACT Currently, diagnosis of acute hepatitis E virus (HEV) in patients is primarily based on anti-HEV immunoglobulin M (IgM) detection. However, several investigations suggest the use of HEV-specific IgA for diagnosing acute HEV infections. We evaluated two commercially available assays, an IgA enzyme-linked immunosorbent assay (ELISA) (Diacheck) and an adapted immunoblot protocol (Mikrogen) for IgA detection and compared the performance in genotype 1- and 3-infected patients. The specificity of the IgA assays was high, with no positive reactions in a control group of 18 acute hepatitis patients who were negative for HEV. The sensitivity calculated in nine PCR-positive type 1-infected patients was 100% in both assays but was clearly lower in genotype 3-infected patients (n = 14), with sensitivities of only 67% and 57% for the ELISA and immunoblot assay, respectively. The lower IgA responses detected in genotype 3-infected patients could be caused by the use of only the genotype 1 and 2 antigens in the serological assays. Interestingly in two patients with possible infection through blood transfusion no response or intermediate IgA responses were detected, and this might confirm the parenteral route of transmission. In both the type 1- and type 3-infected patients both the IgA and IgM responses disappeared simultaneously. We conclude that IgA detection is of limited value for the serodiagnosis of acute HEV cases, particularly with genotype 3.

scholarly journals Use of Serological Assays for Diagnosis of Hepatitis E Virus Genotype 1 and 3 Infections in a Setting of Low Endemicity

2007 ◽  
Vol 14 (5) ◽  
pp. 562-568 ◽  
Author(s):  
M. Herremans ◽  
J. Bakker ◽  
E. Duizer ◽  
H. Vennema ◽  
M. P. G. Koopmans
Keyword(s):  
Hepatitis E Virus ◽  
Specific Antigen ◽  
Hepatitis E ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Genotype 1 ◽  
Genotype 3 ◽  
Virus Genotype ◽  
Immunoblot Assay ◽  
Specificity And Sensitivity

ABSTRACT Because of the occurrence of genotype 3 hepatitis E virus (HEV) in regions of low endemicity, it is important to validate the currently used serological assays for diagnosing infections with viruses belonging to this lineage, since these assays only use antigens derived from genotype 1 and 2 viruses. We evaluated the Genelabs enzyme-linked immunosorbent assay (ELISA) and the RecomBlot from Mikrogen for the detection of HEV-specific immunoglobulin M (IgM) and IgG under conditions of low endemicity. We compared test results of 16 patients with locally acquired genotype 3 HEV, 8 genotype 1 patients, 167 healthy controls from the general population, and 101 cases with hepatitis due to other viral causes. The measured specificities of the ELISA (98%) and the RecomBlot (97%) were comparable to those given by the manufacturer for IgM but were significantly lower for IgG (93% by ELISA and 66% by immunoblotting, versus reported values of 98% for ELISA and 95% for blotting). Antibody levels detected following infections with genotype 3 were lower than those following genotype 1 infections except for those measured in the IgM ELISA. Reactivity to the four antigens used in the immunoblot assay were analyzed and showed differences in the IgM immunoblot reactions between genotype 1 patients and genotype 3 patients. The ORF3 antigen was the most specific antigen. The specificity could be improved by a combined testing regimen with confirmation by immunoblotting of all positive ELISA results and by raising the cutoff of the IgG immunoblot assay without loss of sensitivity. We conclude that a combination of ELISA and immunoblotting is needed for acceptable specificity and sensitivity of HEV assays under conditions of low endemicity.

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

2019 ◽  
Vol 8 (1) ◽  
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.

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

Virulence ◽  
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

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

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