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Journal ArticleDOI

Purification and diagnostic utility of a recombinant hepatitis E virus capsid protein expressed in insect larvae

TL;DR: The expression and purification of a truncated form of the hepatitis E virus ORF2 protein, from the fat bodies of Spodoptera litura larvae infected with a recombinant baculovirus, presents a rapid and low-cost method that obviates the need for expensive tissue culture scale-ups or special equipment.
About: This article is published in Protein Expression and Purification.The article was published on 2003-01-01. It has received 30 citations till now. The article focuses on the topics: Hepatitis E virus & Capsid.
Citations
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Journal ArticleDOI
TL;DR: Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
Abstract: Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

954 citations

Journal ArticleDOI
TL;DR: It is demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells, indicating that a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.
Abstract: The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an ∼56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.

184 citations


Additional excerpts

  • ...(Invitrogen, Carlsbad, CA) as described earlier (57)....

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Journal ArticleDOI
TL;DR: Improved validation of existing anti-HEV antibody assays or development of new assays with superior performance characteristics is urgently needed, as their use for case diagnosis in areas with low endemicity and for seroprevalence studies remains problematic.
Abstract: Hepatitis E, caused by infection with hepatitis E virus (HEV), is a common cause of enterically-transmitted acute hepatitis in developing countries. Occasional cases of sporadic hepatitis E have been increasingly recognized in developed countries over the past decade. These cases differ from those in developing countries in being possibly caused by zoonotic transmission, often affecting people with a suppressed immune system and occasionally leading to persistent HEV infection. The commonly used tests for HEV infection include detection of IgM and IgG anti-HEV antibodies and detection of HEV RNA. IgM anti-HEV antibodies can be detected during the first few months after HEV infection, whereas IgG anti-HEV antibodies represent either recent or remote exposure. The presence of HEV RNA indicates current infection, whether acute or chronic. Although several diagnostic assays for anti-HEV antibodies are available, they have undergone fairly limited testing and often provide discordant results, particularly for IgG antibodies. Thus, although the available antibody assays might be useful for case diagnosis in areas with high disease endemicity, their use for case diagnosis in areas with low endemicity and for seroprevalence studies remains problematic. Improved validation of existing anti-HEV antibody assays or development of new assays with superior performance characteristics is urgently needed.

133 citations

Journal ArticleDOI
TL;DR: When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains, though the involvement of non-cysteine proteases could not be be ruled out.
Abstract: Background: The ORF1 of hepatitis E virus (HEV) encodes a nonstructural polyprotein of ~186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an ~186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system. Results: The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this ~192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT), the first putative domain of the ORF1 polyprotein. Antibodies to the MeT regi on also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag. Conclusion: When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s) could not be be ruled out, this processing mainly depended upon a cysteine protease.

78 citations


Cites methods from "Purification and diagnostic utility..."

  • ...Recombinant virus was harvested by collecting the medium and subsequently used for two rounds of plaque purification followed by the recombinant virus amplification as described earlier [31]....

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Journal ArticleDOI
TL;DR: There is an urgent need to develop better assays for markers of HEV infection and exposure, with low concordance, making comparisons of seroprevalence rates using different assays difficult to interpret.
Abstract: Infection with hepatitis E virus (HEV), mostly genotype 1, is endemic in several developing countries. It can be asymptomatic, be associated with acute hepatitis or fulminant hepatic failure, or present as acute-on-chronic liver disease. Pregnant women are at particular risk of serious outcomes and death. Chronic HEV infection has not been reported from these areas. Diagnosis of acute hepatitis E depends on detection of IgM anti-HEV antibodies or HEV RNA. Current assays for IgM anti-HEV are suboptimal, with high rates of interassay discordance. Though they perform reasonably well in disease-endemic areas, positive test results in low-endemicity areas require confirmation using HEV RNA testing. Detection of IgG anti-HEV antibodies indicates exposure to HEV, either recent or remote. Assays for these too have low concordance, making comparisons of seroprevalence rates using different assays difficult to interpret. There is an urgent need to develop better assays for markers of HEV infection and exposure.

64 citations

References
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Journal ArticleDOI
TL;DR: Intravenous inoculation of cynomolgus monkeys with the virus-containing stool extract resulted in histopathologically and enzymatically confirmed hepatitis, excretion of virus-like particles, and antibody response to them.
Abstract: Typical acute hepatitis was reproduced in a human volunteer immune to hepatitis A virus (HAV) after oral administration of pooled stool extracts from presumed cases of epidemic non-A, non-B hepatitis. Markers of hepatitis B infection, anti-HAV IgM, and increase in total anti-HAV level were not detectable in the volunteer’s sera during the course of infection. Spherical 27- to 30-nm virus-like particles were visualized by immune electron microscopy (IEM) in stool samples collected during preclinical and early postclinical phases. These particles banded in CsCl at a buoyant density of 1.35 g/cm3. They reacted in the IEM test with sera from individuals who had experienced two non-B hepatitis episodes but did not react with sera from routine anti-HAV IgM-positive hepatitis patients. Intravenous inoculation of cynomolgus monkeys with the virus-containing stool extract resulted in histopathologically and enzymatically confirmed hepatitis, excretion of virus-like particles, and antibody response to them.

767 citations

Journal ArticleDOI
13 Jun 1985-Nature
TL;DR: A virus vector to introduce foreign genes, for example, the gene for human α-Interferon (IFN-α), into silkworms is developed, which has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field.
Abstract: Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.

495 citations

Journal ArticleDOI
TL;DR: It is found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column.
Abstract: Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.

403 citations

Journal ArticleDOI
01 Dec 1992-Virology
TL;DR: Knowing the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further the understanding of how HEV strain variation might impact the pathological outcome of infection.

326 citations