scholarly journals Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

2011 ◽  
Vol 92 (12) ◽  
pp. 2830-2837 ◽  
Author(s):  
Tian-Cheng Li ◽  
Kumiko Yoshimatsu ◽  
Shumpei P. Yasuda ◽  
Jiro Arikawa ◽  
Takaaki Koma ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Hepatitis E ◽  
Serum Samples ◽  
Electron Microscopic ◽  
Rat Serum ◽  
Wild Rats ◽  
Self Assembled

Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

scholarly journals Characterization of self-assembled virus-like particles of ferret hepatitis E virus generated by recombinant baculoviruses

2013 ◽  
Vol 94 (12) ◽  
pp. 2647-2656 ◽  
Author(s):  
Tingting Yang ◽  
Michiyo Kataoka ◽  
Yasushi Ami ◽  
Yuriko Suzaki ◽  
Noriko Kishida ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Hepatitis E ◽  
Electron Microscopic ◽  
Antigenic Analysis ◽  
Virus Like Particles ◽  
C Terminus ◽  
N Terminus ◽  
Self Assembled

Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in The Netherlands. Due to the lack of a cell-culture system for ferret HEV, the antigenicity, pathogenicity and epidemiology of this virus have remained unclear. In the present study, we used a recombinant baculovirus expression system to express the 112-N-terminus and 47-C-terminus-amino-acid-truncated ferret HEV ORF2 protein in insect Tn5 cells, and found that a large amount of a 53 kDa protein (F-p53) was expressed and efficiently released into the supernatant. Electron microscopic analysis revealed that F-p53 was self-assembled into virus-like particles (ferret HEV-LPs). These ferret HEV-LPs were estimated to be 24 nm in diameter, which is similar to the size of G1, G3, G4 and rat HEV-LPs derived from both the N-terminus- and C-terminus-truncated constructs. Antigenic analysis demonstrated that ferret HEV-LPs were cross-reactive with G1, G3, G4 and rat HEVs, and rat HEV and ferret HEV showed a stronger cross-reactivity to each other than either did to human HEV genotypes. However, the antibody against ferret HEV-LPs does not neutralize G3 HEV, suggesting that the serotypes of these two HEVs are different. An ELISA for detection of anti-ferret HEV IgG and IgM antibodies was established using ferret HEV-LPs as antigen, and this assay system will be useful for monitoring ferret HEV infection in ferrets as well as other animals. In addition, analysis of ferret HEV RNA detected in ferret sera collected from a breeding colony in the USA revealed the genetic diversity of ferret HEV.

scholarly journals Cloning and expression of hepatitis E virus ORF2 as an immunogen protein in baculovirus expression system

2018 ◽  
Vol 5 (1) ◽  
pp. 23-26
Author(s):  
SA Sadeghi ◽  
M Shahanaghi ◽  
MR Aghasadeghi ◽  
F Motevalli ◽  
MR Amiran ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Hepatitis E ◽  
Cloning And Expression ◽  
Baculovirus Expression

scholarly journals Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

2010 ◽  
Vol 43 (3) ◽  
pp. 229-233 ◽  
Author(s):  
Alex Martins Machado ◽  
Glauciane Garcia Figueiredo ◽  
Gelse Maria Campos ◽  
Mario Enrique Lozano ◽  
Aline Rafaela da Silva Rodrigues Machado ◽  
...  
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Hemorrhagic Fever ◽  
Baculovirus Expression System ◽  
Elisa Test ◽  
Serum Samples ◽  
Serological Screening ◽  
Mato Grosso ◽  
Junin Virus ◽  
Junín Virus

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

scholarly journals The First Documented Detection in Taiwan of The Hepatitis E Virus in Rats

2020 ◽  
Author(s):  
Pai-Shan Chiang ◽  
Wei-Lun Huang ◽  
Han-Hsuan Chung ◽  
Jyh-Yuan Yang ◽  
Hwa-Jen Teng
Keyword(s):  
Rattus Rattus ◽  
Hepatitis E ◽  
Human Infection ◽  
Serum Samples ◽  
Rat Serum ◽  
Negative Results ◽  
Human Sera ◽  
Wild Rats ◽  
Human Infections ◽  
Rodent Populations

Abstract Background: Human infections by rat HEV (HEV-C1) have been serially reported, including a case who had visited Taiwan before having the illness in 2019. The objective of this study was to investigate whether HEV-C1 is circulating and causing human infections in Taiwan.Methods: Fifty acute-phase human sera samples from HEV suspected patients with the negative results were randomly chosen for retrospective review. Rat sera were collected from 3 Rattus rattus and 47 R. norvegicus, which were captured at international airports or harbors. Identifying HEV-C1 RNA was performed by hemi-nested RT-PCR in human and rat serum samples. Rat sera were also tested for anti-rat HEV antibodies. Results: HEV-C1 RNA was not detected in either human or R. rattus samples, but the viral RNA was identified in two R. norvegicus samples. The 2 rat HEV strains shared identical partial sequences in the RNA polymerases gene. In serology, anti-HEV antibodies were detected in 52% (26/50) of the trapped wild rats.Conclusions: This study documents the first detection of HEV-C1 in Taiwan. The high homology between HEV-C1 sequences from rats observed in this study might result from viral circulation and transmission within certain rodent populations. The risk of indigenous human infection in Taiwan should not be ignored because of the domestic detection of HEV-C1 RNA.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Serological and molecular studies on subclinical hepatitis E virus infection using periodic serum samples obtained from healthy individuals

2005 ◽  
Vol 76 (4) ◽  
pp. 526-533 ◽  
Author(s):  
Takehiro Mitsui ◽  
Yukie Tsukamoto ◽  
Shigeru Suzuki ◽  
Chikao Yamazaki ◽  
Kazuo Masuko ◽  
...  
Keyword(s):  
Virus Infection ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Healthy Individuals ◽  
Serum Samples ◽  
Molecular Studies

Seroprevalence of the hepatitis E virus antibodies among blood donors in the Qassim region, Saudi Arabia

2021 ◽  
Author(s):  
Bader Y Alhatlani ◽  
Waleed A Aljabr ◽  
Mohammed S Almarzouqi ◽  
Sami M Alhatlani ◽  
Rayan N Alzunaydi ◽  
...  
Keyword(s):  
Public Health ◽  
Saudi Arabia ◽  
Blood Transfusion ◽  
Hepatitis E Virus ◽  
Blood Donors ◽  
Indirect Elisa ◽  
Hepatitis E ◽  
Public Health Issue ◽  
Serum Samples ◽  
Major Public Health Issue

Aim: Hepatitis E virus (HEV) transmission through blood transfusion is a major public health issue worldwide. We aimed to determine the seroprevalence of HEV in blood donors in the Qassim region of Saudi Arabia. Materials & methods: Serum samples (n = 1078) were collected from volunteer blood donors and tested for the presence of anti-HEV IgG and IgM by indirect ELISA. Results: The seroprevalence of anti-HEV IgG among the blood donors was 5.7% overall. Anti-HEV IgG and IgM seropositivity were significantly higher in non-Saudi donors than in Saudi donors (22.1 vs 3 and 7.8 vs 0.2% for anti-HEV IgG and IgM, respectively). Conclusion: The seroprevalence of HEV among blood donors in the Qassim region was lower than previous estimates for other regions of the country and neighboring countries.

scholarly journals Diagnostic Performance of Five Assays for Anti-Hepatitis E Virus IgG and IgM in a Large Cohort Study

2015 ◽  
Vol 54 (3) ◽  
pp. 549-555 ◽  
Author(s):  
Heléne Norder ◽  
Marie Karlsson ◽  
Åsa Mellgren ◽  
Jan Konar ◽  
Elisabeth Sandberg ◽  
...  
Keyword(s):  
Gold Standard ◽  
Diagnostic Performance ◽  
Hepatitis E Virus ◽  
Blood Donors ◽  
Laboratory Diagnosis ◽  
Hepatitis E ◽  
Serum Samples ◽  
Large Cohort Study ◽  
No Gold Standard

Determination of anti-hepatitis E virus (anti-HEV) antibodies is still enigmatic. There is no gold standard, and results obtained with different assays often diverge. Herein, five assays were compared for detection of anti-HEV IgM and IgG. Serum samples from 500 Swedish blood donors and 316 patients, of whom 136 had suspected HEV infection, were analyzed. Concordant results for IgM and IgG with all assays were obtained only for 71% and 70% of patients with suspected hepatitis E, respectively. The range of sensitivity for anti-HEV detection was broad (42% to 96%); this was reflected in the detection limit, which varied up to 19-fold for IgM and 17-fold for IgG between assays. HEV RNA was analyzed in all patients and in those blood donors reactive for anti-HEV in any assay, and it was found in 26 individuals. Among all of the assays, both anti-HEV IgG and IgM were detected in 10 of those individuals. Twelve had only IgG and, in 7 of those 12, IgG was only detected with the two most sensitive assays. Three of the HEV-RNA-positive samples were negative for anti-HEV IgM and IgG in all assays. With the two most sensitive assays, anti-HEV IgG was identified in 16% of the blood donor samples and in 66% of patients with suspected HEV infection. Because several HEV-RNA-positive samples had only anti-HEV IgG without anti-HEV IgM or lacked anti-HEV antibodies, analysis for HEV RNA may be warranted as a complement in the laboratory diagnosis of ongoing HEV infection.

Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies

2014 ◽  
Vol 95 (10) ◽  
pp. 2166-2175 ◽  
Author(s):  
Shigeo Nagashima ◽  
Suljid Jirintai ◽  
Masaharu Takahashi ◽  
Tominari Kobayashi ◽  
Tanggis ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Lipid Membrane ◽  
Hepatitis E ◽  
Negative Control ◽  
Bafilomycin A1 ◽  
Genotype 3 ◽  
Electron Microscopic ◽  
Immunogold Staining ◽  
Virus Particles ◽  
Virion Release

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

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