Detection of Cell Membrane Proteins that Interact with Virulent Infectious Bursal Disease Virus
Abstract and Figures
To detect the molecules that interact with infectious bursal disease virus (IBDV), the chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for virulent IBDV infection was investigated. The sodium dodecyl sulfate-solubilized plasma membrane fraction from the cells was subjected to a virus overlay protein binding assay. The IBDV specifically bound to proteins in LSCC-BK3 plasma membranes with molecular weights of 70, 82 and 110 kDa. This is the first report to demonstrate cellular molecules that interact with virulent IBDV.
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... It was suggested that the IBDV host range is mainly controlled by a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by sIgM bearing cells. Setiyono et al. (2001) conducted a study to detect the molecules that interact with IBDV. They subjected SDS-solubilized plasma membrane fraction from LSCC-BK3 cells to a virus overlay protein-binding assay. ...
Gangale D P and Saxena H M. (2008). Host-pathogen interaction in infectious bursal disease of chickens. J. Immunol. Immunopathol. 10(2): 98-104. Infectious Bursal Disease (IBD) is an acute contagious disease of young chickens with high morbidity and low mortality causing heavy losses to poultry industry worldwide. The disease is caused by IBD virus (IBDV), an Avibirnavirus which infects immature bursal B-cells of young chicken. However, the target molecule of B cells for binding of IBDV is not known. This review attempts to discuss the various possibilities and the current research in this direction.
... It was suggested that the IBDV host range is mainly controlled by a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by sIgM bearing cells. Setiyono et al. (2001) conducted a study to detect the molecules that interact with IBDV. They subjected SDS-solubilized plasma membrane fraction from LSCC-BK3 cells to a virus overlay protein-binding assay. ...
Gangale D P and Saxena H M. (2008). Host-pathogen interaction in infectious bursal disease of chickens. J. Immunol. Immunopathol. 10(2): 98-104. Infectious Bursal Disease (IBD) is an acute contagious disease of young chickens with high morbidity and low mortality causing heavy losses to poultry industry worldwide. The disease is caused by IBD virus (IBDV), an Avibirnavirus which infects immature bursal B-cells of young chicken. However, the target molecule of B cells for binding of IBDV is not known. This review attempts to discuss the various possibilities and the current research in this direction.
... The residues Gln 253, Asp 279 and Ala 384 of VP2 of IBDV are known to be involved in the virulence, cell tropism and pathogenic phenotypes of virulent IBDV (Brandt et al., 2001). Setiyono et al. (2001) generated three hybridoma cell lines producing monoclonal antibodies (mAbs) against LSCC-BK3 cells. The mAbs designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. ...
Gangale D P and Saxena H M. (2007). Inhibition of binding of Infectious Bursal Disease Virus to chicken bursal B-cells by treatment of B-cells with antibody against a marker of immature B-cells. J. Immunol. Immunopathol. 9(1 & 2): 50-53. Infectious Bursal Disease (IBD) is an important disease of young chickens having significant economic implications for poultry industry worldwide. The exact target molecule for attachment of IBD virus (IBDV) to chicken B-cells is not known. Three monoclonal antibodies (T7, Q11 and Q13) against markers of immature B-cells of chicken were used to treat the bursal B-cells of young chickens (6 weeks old) before incubating them with IBDV. Immunoperoxidase staining of IBDV was used to assess the level of IBDV binding to B-cells without or after treatment with any of the three antibodies. The percentages of IBDV positive B-cells without and after treatment of B-cells with the T7, Q11 and Q13 antibodies were 93.42±0.47, 89.42±0.69, 91.75±0.35 and 91.58±0.57, respectively. The differences of mean values between the groups without antibody treatment and after antibody treatment were very significant in case of Q11 and Q13 antibodies (p = 0.001) and extremely significant (p<0.0001) in case of T7 antibody. ANOVA revealed an extremely significant (F = 9.624, p<0.0001) difference among the control and the three antibodies. A comparison of means between the various groups by Tukey's HSD test revealed significant differences between the control group and the three antibodies. The results suggest that the antibody T7 may be recognizing some epitope which may possibly be the target or located on or adjacent to the site of IBDV binding on B-cells.
... It was suggested that the IBDV host range is mainly controlled by a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by sIgM bearing cells. Setiyono et al. (2001) conducted a study to detect the molecules that interact with IBDV. They subjected SDS-solubilized plasma membrane fraction from LSCC-BK3 cells to a virus overlay protein-binding assay. ...
... The very virulent IBDV strains are unable to propagate directly in tissue culture and attenuation by repeated passages of vvIBDV in chicken embryonic fibroblasts (Yamaguchi et al., 1996a,b) and Vero cells (Kwon et al., 2000) is necessary for tissue culture adaptation. There is, however, one study which showed a vvIBDV propagated in LSCC-BK3 cells without adaptation (Setiyono et al., 2001). Attenuated strains are distinguished by residues His 253 and Ala 284 in place of Gln 253 and Thr 284 found in the other strains. ...
... Virus overlay protein blotting assay (VOPBA) is a useful method to study the interaction between virus and receptor in vitro (Kim et al., 2006). Using the method Nieper et al. (1996) and Setiyono et al. (2001) detected the molecules showing properties of binding to IBDV particles , which were expressed on CEF cells and LSCC-BK3, respectively. Nieper found that proteins with molecular mass of 40 and 46 kDa expressed in both CEF cells and chicken B lymphocytes could bind to the viral particles propagated in CEF cells. ...
Virus receptors are simplistically defined as cell surface molecules that mediate binding (attachment, adsorption) and/or trigger membrane fusion or entry through other processes. Infectious bursal disease virus (IBDV) entry into host cells occurs by recognition of specific cellular receptor(s) with viral envelope glycoprotein, which comprises the initial and key step of infection. Infection can be inhibited by blockage of the process. So the interest in receptors has been stimulated in large part by the potential in the application of developing substances that show directed blocking activity. While for the purpose one should know which host cell and viral molecules are involved in the reciprocal recognition and interaction leading to the virus entry into the cell. Here, the review presents the currently available knowledge regarding the receptors or molecules that interact with IBDV.
... The load and replication of IBDV in the bursa is closely related to clinical symptoms and pathology (Eldaghayes et al., 2006;Liu et al., 2010). The major targets of IBDV are surface immunoglobulin M (sIgM)-bearing B lymphocytes (Setiyono et al., 2001;Rodr ıguez-Lecompte et al., 2005). Petkov et al. (2009) identified two different IgM B-cell subpopulations that are designated as A and B. The authors suggest that subpopulation A appeared to be resistant to IBDV challenge, remained at constant levels regardless of the challenge or vaccination and may be at an earlier developmental stage. ...
Humoral immune responses in birds, contrary to mammals, depend on the normal functioning of bursa Fabricii. Recent studies have delivered new information about the structure, development and origin of cells that compose the bursa environment. Several viral infections affect bursa, causing lymphocyte depletion or excessive proliferation. This review summarizes data on the development and histology of healthy bursa and introduces some common disorders that affect this organ.
Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.
Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. The identification of host cellular molecules that bind to IBDV will improve the understanding of the underlying pathogenic mechanisms. In this study, using a virus overlay protein-binding assay (VOPBA) and mass spectrometry (MS) analysis, IBDV was found to bind chicken Anx2, a membrane protein fraction from DF-1 cells. Its interactions were further confirmed by an overlay assay. The results of an immunofluorescence assay and flow cytometry showed that Anx2 could be expressed and colocalized with IBDV on the surface of infected cells. Moreover, either the soluble recombinant Anx2 or an anti-Anx2 antibody could inhibit IBDV binding to and infection of DF-1 cells in a dose-dependent manner. The knockdown of Anx2 of DF-1 cells by small interfering RNA clearly reduced the subsequent virus yield, and overexpression of Anx2 was capable of enhancing the virus yield. These results indicate, for the first time, that binding to Anx2 is beneficial for IBDV infection.
Copyright © 2015. Published by Elsevier B.V.
The molecular mechanism of genetic resistance of inbred mouse strains to mouse hepatitis virus, a murine coronavirus, was studied by comparing virus binding to plasma membranes of intestinal epithelium or liver from susceptible BALB/c and resistant SJL/J mice with a new solid-phase assay for virus-binding activity. Virus bound to isolated membranes from susceptible mice, but not to membranes from resistant mice. F1 progeny of SJL/J X BALB/c mice had an intermediate level of virus-binding activity on their enterocyte and hepatocyte membranes. This correlated well with previous studies showing that susceptibility to mouse hepatitis virus strain A59 is controlled by a single autosomal dominant gene (M. S. Smith, R. E. Click, and P. G. W. Plagemann, J. Immunol. 133:428-432). Because virus binding was not prevented by treating membranes with sodium dodecyl sulfate, the virus-binding molecule could be identified by a virus overlay protein blot assay. Virus bound to a single broad band of Mr 100,000 to 110,000 in membranes from hepatocytes or enterocytes of susceptible BALB/c and semisusceptible C3H mice, but no virus-binding band was detected in comparable preparations of resistant SJL/J mouse membranes. Therefore, SJL/J mice may be resistant to mouse hepatitis virus A59 infection because they lack a specific virus receptor which is present on the plasma membranes of target cells from genetically susceptible BALB/c and semisusceptible C3H mice.
The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 X 10(6). The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one of the established taxonomic groups.
A naturally acquired subclinical infection of infectious bursal agent was shown to be the probable causative factor of marked immunosuppression of Newcastle disease vaccine in young chicks. Old hens maintained in the same contaminated environment did not exhibit immunosuppression.
Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.
The in vitro susceptibility of chicken lymphocytes to a wild strains of infectious bursal disease virus was investigated by using immunofluorescence and virus assays as infection criteria. A variety of Marek's disease lymphoblastoid cell lines, all of thymus (T-cell) origin, were refractory to virus exposure. However, a bursa (B-cell)-derived lymphoblastoid cell line from an avian leukosis virus-induced tumor was highly susceptible. Viral antigen appeared in the cytoplasm of 20 to 30% of the cells, and large amounts of cell-free virus were released, with maximum yields occurring by 3 days postinfeciton. The virus also replicated in a small percentage of normal lymphocytes prepared from lymphoid tissues and peripheral blood of chickens. Pretreatment of the lymphocytes, with heat-inactivated anti-B-cell serum or with antiserum against fowl immunoglobulin M before inoculating them with the virus blocked the virus infection; no blocking occurred with anti-T-cell serum or with specific antiserum against fowl immunoglobulin G or immunoglobulin A. This suggests that surface immunoglobulin M-bearing B-lymphocytes were the target cells for infection.
The attachment of lymphocytic choriomeningitis virus (LCMV) to murine and primate cell lines was quantitated by a fluorescence-activated cell sorter assay in which binding of biotinylated virus was detected with streptavidin-fluorescein isothiocyanate. Cell lines that were readily infected by LCMV (e.g., MC57, Rin, BHK, Vero, and HeLa) bound virus in a dose-dependent manner, whereas no significant binding was observed to lymphocytic cell lines (e.g., RMA and WIL 2) that were not readily infected. Binding was specific and competitively blocked by nonbiotinylated LCMV. It was also blocked by LCMV-specific antiserum and a neutralizing monoclonal antibody to the virus glycoprotein GP-1 but not by antibodies specific for GP-2, indicating that attachment was likely mediated by GP-1. Treatment of cells with any of several proteases abolished LCMV binding, whereas phospholipases including phosphatidylinositol-specific phospholipase C had no effect, indicating that one or more membrane proteins were involved in virus attachment. These proteins were characterized with a virus overlay protein blot assay. Virus bound to protein(s) with a molecular mass of 120 to 140 kDa in membranes from cell lines permissive for LCMV but not from nonpermissive cell lines. Binding was specific, since unlabeled LCMV, but not the unrelated enveloped virus herpes simplex virus type 1, competed with 125I-labeled LCMV for binding to the 120- to 140-kDa band. The proteinaceous nature of the LCMV-binding substance was confirmed by the lack of virus binding to proteinase K-treated membrane components. By contrast, glycosidase treatment of membranes did not abolish virus binding. However, in membranes treated with endoglycosidase F/N-glycosidase F, and/or neuraminidase and in membranes from cells grown in tunicamycin, the molecular mass of the LCMV-binding entity was reduced. Hence, LCMV attachment to rodent fibroblastic cell lines is mediated by a glycoprotein(s) with a molecular mass of 120 to 140 kDa, with complex N-linked sugars that are not involved in virus binding.
Infectious bursal disease virus of chickens was purified, and its structure was examined by the negative-staining technique in the electron microscope. The buoyant density of infectious bursal disease virus in CsCl was found to be 1.34 g/cm(3). The morphological details suggest that the capsid of the virion consists of a single layer of 32 capsomeres arranged in 5:3:2 symmetry. The virion measured about 55 nm in diameter and had no envelope.
Infectious bursal disease virus (IBVD) depressed the humoral antibody response of chickens to various vaccines. The virus had that effect whether injected at the same time as vaccination or 4 or 7 days before. The depression was slightly greater when injection was 7 days before, and was most pronounced in chickens inoculated with IBDV when 6 wk old, slight in 4 wk olds, and absent in 2 wk olds. Protection against challenge by virulent ND virus 3 wk after ND vaccination showed the marked differences between IBDV inoculated and uninoculated chickens.
The infectious bursal disease virus (IBDV) was adapted to chicken embryo bursal (CEB) and kidney (CEK) cells. The virus did not replicate in the kidney cells until 4 serial passages of the virus were made in bursal cells. The virus produced plaques in CEK cells in 3 to 5 days. The replication of the virus was unaltered by 5 iodo 2' deoxyuridine, indicating that it is a ribovirus. The infectivity of the virus was not destroyed with chloroform and was stable at 56 C for 90 min. The virus attached to the CEK cell monolayer maximally in 75 min and had an eclipse period of 9 to 10 hours before virus maturation and release. Immunofluorescent studies of infected cells showed that the first antigen was detected 6 hr postinfection and all fluorescing antigen was confined to the cytoplasm. All of these characteristics lend support to the classification of IBDV in the diplornavirus group of riboviruses.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.