Characterization of recombinant norovirus virus-like particles and evaluation of their applicability to the investigation of norovirus removal performance in membrane filtration processes

2015 ◽  
Vol 16 (3) ◽  
pp. 737-745
Author(s):  
N. Shirasaki ◽  
T. Matsushita ◽  
Y. Matsui ◽  
K. Ohno
Keyword(s):  
Membrane Filtration ◽  
Expression System ◽  
Small Animal ◽  
Microscopic Analysis ◽  
Peptide Mass Fingerprinting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Electron Microscopic ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Peptide Mass

Noroviruses (NVs) are one of the leading causes of epidemic gastroenteritis around the world. Water treatment technologies using membrane filtration for virus removal are becoming increasingly important. However, experiments to test removal of NVs from water have been hampered because NVs do not grow in cell culture or in small-animal models and therefore cannot be easily artificially propagated. Expression of the NV genome in a baculovirus-silkworm expression system has produced recombinant NV virus-like particles (rNV-VLPs) that are morphologically and antigenically similar to native NV. Here, we characterized these rNV-VLPs and evaluated their potential use in assessing NV removal. Electron microscopic analysis and peptide mass fingerprinting showed that the rNV-VLPs were morphologically identical to native NV. In addition, surface charge and particle size distribution, which are important factors for explaining virus particle behavior during membrane filtration, were successfully evaluated by using rNV-VLPs. The rNV-VLPs were easy to quantify with a commercially available enzyme-linked immunosorbent assay kit, they remained stable for several days at 4 °C after dilution in river water, and they were easy to concentrate with the ultrafiltration entrapment method used. Thus, rNV-VLPs can be used to facilitate our understanding of the behavior of NVs during membrane filtration processes.

scholarly journals Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

2004 ◽  
Vol 70 (7) ◽  
pp. 3904-3909 ◽  
Author(s):  
Santiago Caballero ◽  
F. Xavier Abad ◽  
Fabienne Loisy ◽  
Françoise S. Le Guyader ◽  
Jean Cohen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Uv Light ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Reverse Transcription Pcr ◽  
Actual Field ◽  
Uv Dose ◽  
Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

scholarly journals Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

2002 ◽  
Vol 76 (15) ◽  
pp. 7672-7682 ◽  
Author(s):  
Reginald F. Clayton ◽  
Ania Owsianka ◽  
Jim Aitken ◽  
Susan Graham ◽  
David Bhella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Recombinant Baculovirus ◽  
Microscopic Analysis ◽  
Surface Topology ◽  
Viral Glycoprotein ◽  
Electron Microscopic ◽  
Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

Specific antibodies to moesin, a membrane-cytoskeleton linker protein, are frequently detected in patients with acquired aplastic anemia

Blood ◽  
2006 ◽  
Vol 109 (6) ◽  
pp. 2514-2520 ◽  
Author(s):  
Hiroyuki Takamatsu ◽  
Xingmin Feng ◽  
Tatsuya Chuhjo ◽  
Xuzhang Lu ◽  
Chiharu Sugimori ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Leukemia Cell ◽  
Peptide Mass Fingerprinting ◽  
Leukemia Cell Line ◽  
Enzyme Linked Immunosorbent Assay ◽  
Membrane Cytoskeleton ◽  
Peptide Mass ◽  
Leukemia Cell Lines ◽  
Culture Supernatants ◽  
Myeloid Leukemia Cell

Abstract To identify novel autoantibodies in acquired aplastic anemia (AA), we screened the sera of patients with AA possessing small populations of paroxysmal nocturnal hemoglobinuria (PNH)–type cells for the presence of antibodies (Abs) which recognize proteins derived from a leukemia cell line, UT-7. Immunoblotting using proteins derived from lysates or culture supernatants of UT-7 cells revealed the presence of IgG Abs specific to an 80-kDa protein. Peptide mass fingerprinting identified this 80-kDa protein as moesin. Enzyme-linked immunosorbent assay (ELISA) using recombinant moesin showed high titers of antimoesin Abs in 25 (37%) of 67 patients with AA. Moesin was secreted from several myeloid leukemia cell lines other than UT-7, such as OUN-1 and K562, as an exosomal protein. The presence of antimoesin Abs was significantly correlated with the presence of PNH-type cells and antidiazepam-binding inhibitor-related protein-1 (DRS-1) Abs. Patients with AA that did not show any of these 3 markers tended to respond poorly to immunosuppressive therapy. These findings suggest that a B-cell response to moesin, possibly derived from hematopoietic cells, frequently occurs in patients with AA and that detection of antimoesin Abs in combination with other markers may be useful in diagnosing immune pathophysiology in patients with AA.

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 First Report of Celery mosaic virus Infecting Celery in Venezuela

Plant Disease ◽  
2006 ◽  
Vol 90 (8) ◽  
pp. 1111-1111 ◽  
Author(s):  
T. Fernández ◽  
O. Carballo ◽  
K. Zambrano ◽  
M. Romano ◽  
E. Marys
Keyword(s):  
Mosaic Virus ◽  
Microscopic Analysis ◽  
Apium Graveolens ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Leaf Extracts ◽  
Electron Microscopic ◽  
First Report ◽  
Three Samples ◽  
Topo Vector

Celery mosaic virus (CeMV) is a significant pathogen of celery (Apium graveolens) worldwide (1). In 2005, in a produce market located in Los Salias, Miranda, celery plants with mottling and leaf malformation were noticed. Electron microscopic analysis of leaf-dip preparations from three symptomatic samples revealed flexuous viral particles that were 750 nm long. Infected cells contained pinwheel inclusions typical of those associated with potyvirus infection. Inoculation of healthy celery plants with leaf extracts from four symptomatic plants produced symptoms identical to those first observed. A survey of five produce markets in Miranda was conducted to determine the prevalence of virus infection in celery using serological and molecular analyses. Mottling and malformation of celery leaflets were observed in all the markets visited. Symptoms were noted in all five markets in each of three visits during a 3-month period. A total of 125 postharvested symptomatic plants were collected from five markets on March 29, 2005 and tested for CeMV using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with antiserum provided by F. Rabenstein, BAX, Aschersleben, Germany. Of the 125 samples collected during the survey, 53% were ELISA positive. Twenty ELISA-positive samples were also tested using reverse transcription-polymerase chain reaction (RT-PCR) with general primers for the family Potyviridae (2). All 20 samples produced an amplicon of the expected size (1.7 kbp) after RT-PCR. Amplicons from three samples were cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA). Sequence analysis of one clone revealed more than 98% nt to a CeMV isolate from Australia (GenBank Accession No AF203532). To our knowledge, this is the first report of CeMV in Venezuela. Our results suggest that the disease may be widely spread on celery crops growing in the areas surrounding produce markets in Miranda State. References: (1) A. Brunt et al. Viruses of Plants. CAB International, Wallingford, Oxon, UK. 1996. (2) J. Chen et al. Arch. Virol. 146:757, 2001.

scholarly journals Development and Validation of A Competitive ELISA Based on Virus-Like Particles of Serotype Senecavirus A to Detect Serum Antibodies

2020 ◽  
Author(s):  
Manyuan Bai ◽  
Rui Wang ◽  
Shiqi Sun ◽  
Yun Zhang ◽  
Hu Dong ◽  
...  
Keyword(s):  
Expression System ◽  
Antibody Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Virus Like Particles ◽  
Specificity And Sensitivity ◽  
Indirect Immunofluorescent Assay ◽  
Viral Particles ◽  
Test Kit ◽  
Development And Validation

Abstract Virus-like particles (VLPs) are high-priority antigens with highly ordered repetitive structures, which are similar to natural viral particles. We have developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies directed against Senecavirus A (SVA). Our assay utilizes SVA VLPs that were expressed and assembled in an Escherichia coli expression system as the coating antigens. VLPs have better safety and immunogenicity than intact viral particles or peptides. The VLP-based cELISA was used to test 342 serum samples collected from different pig farms, and the results showed that its specificity and sensitivity were 100% and 94%, respectively. The consistency rates of cELISA with the BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit and an indirect immunofluorescent assay were 90.0% and 94.2%, respectively. Therefore, this VLP-based cELISA can be effectively and reliably used for the detection and discrimination of SVA infection in serum samples.

scholarly journals Development and validation of a competitive ELISA based on virus-like particles of serotype Senecavirus A to detect serum antibodies

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manyuan Bai ◽  
Rui Wang ◽  
Shiqi Sun ◽  
Yun Zhang ◽  
Hu Dong ◽  
...  
Keyword(s):  
Expression System ◽  
Antibody Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Virus Like Particles ◽  
Specificity And Sensitivity ◽  
Indirect Immunofluorescent Assay ◽  
Viral Particles ◽  
Test Kit ◽  
Development And Validation

AbstractVirus-like particles (VLPs) are high-priority antigens with highly ordered repetitive structures, which are similar to natural viral particles. We have developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies directed against Senecavirus A (SVA). Our assay utilizes SVA VLPs that were expressed and assembled in an E. coli expression system as the coating antigens. VLPs have better safety and immunogenicity than intact viral particles or peptides. The VLPs-based cELISA was used to test 342 serum samples collected from different pig farms, and the results showed that its specificity and sensitivity were 100% and 94%, respectively. The consistency rates of cELISA with the BIOSTONE AsurDx™ Senecavirus A (SVA) Antibody Test Kit and an indirect immunofluorescent assay were 90.0% and 94.2%, respectively. Therefore, this VLPs-based cELISA can be effectively and reliably used for the detection and discrimination of SVA infection in serum samples.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Electron Microscopic Analysis of Myonecrosis Induced by a Pure Component Isolated From Rattlesnake Venom

1976 ◽  
Vol 34 ◽  
pp. 292-293
Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu
Keyword(s):  
Gel Filtration ◽  
Microscopic Analysis ◽  
The United States ◽  
Exchange Chromatography ◽  
Pure Component ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Mouse Muscle ◽  
Major Health Problem ◽  
Rattlesnake Venom

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.

Correlative video enhanced differential interference contrast light microscopy (VDIC)-LM), low-voltage high-resolution SEM (LV-HR-SEM), and high-voltage electron microscopy (HVEM) in the observation of gold-labelled surface antigens and the subjacent cytoskeletal matrix

1989 ◽  
Vol 47 ◽  
pp. 904-905
Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
Marek Malecki
Keyword(s):  
High Resolution ◽  
Light Microscopy ◽  
Low Voltage ◽  
Microscopic Analysis ◽  
Cell Labelling ◽  
Video Technology ◽  
Electron Microscopic ◽  
Image Capture ◽  
High Voltage Electron Microscopy ◽  
Fluorescence Studies

The development of video-enhanced light microscopy (LM) as well as associated image processing and analysis have significantly broadened the scope of investigations which can be undertaken using (LM). Interference/polarization based microscopies can provide high resolution and higher levels of “detectability” especially in unstained living systems. Confocal light microscopy also holds the promise of further improvements in resolution, fluorescence studies, and 3 dimensional reconstruction. Video technology now provides, among other things, a means to detect differences in contrast difficult to detect with the human eye; furthermore, computerized image capture, processing, and analysis can be used to enhance features of interest, average images, subtract background, and provide a quantitative basis to studies of cells, cell features, cell labelling, and so forth. Improvements in video technology, image capture, and cost-effective computer image analysis/processing have contributed to the utility and potential of the various interference and confocal microscopic instrumentation.Electron microscopic technology has made advances as well. Microprocessor control and improved design have contributed to high resolution SEMs which have imaging capability at the molecular level and can operate at a range of accelerating voltages starting at 1KV. Improvements have also been seen in the HVEM and IVEM transmission instruments. As a whole, these advances in LM and EM microscopic technology provide the biologist with an array of information on structure, composition, and function which can be obtained from a single specimen. Corrrelative light microscopic analysis permits examination of living specimens and is critical where the “history” of a cell, cellular components, or labels needs to be known up to the time of chemical or physical fixation. Features such as cytoskeletal elements or gold label as small as 0.01 μm, well below the 0.2 μm limits of LM resolution, can be “detected” and their movement followed by VDIC-LM. Appropriate identification and preparation can then lead to the examination of surface detail and surface label with stereo LV-HR-SEM. Increasing the KV in the HR-SEM while viewing uncoated or thinly coated specimens can provide information from beneath the surface as well as increasing Z contrast so that positive identification of surface and subsurface colloidal gold or other heavy metal labelled/stained material is possible. Further examination of the same cells using stereo HVEM or IVEM provides information on internal ultrastructure and on the relationship of labelled material to cytoskeletal or organellar distribution, A wide variety of investigations can benefit from this correlative approach and a number of instrumentational configurations and preparative pathways can be tailored for the particular study. For a surprisingly small investment in time and technique, it is often possible to clear ambiguities or questions that arise when a finding is presented in the context of only one modality.

Sign in / Sign up

Export Citation Format

Share Document