Rabies Virus Binding Research Articles

Long-term immunity induced by SPBN GASGAS in orally vaccinated dogs is non-inferior to inactivated rabies vaccines

In a long-term immunogenicity study (1100 days post vaccination) in local Thai dogs the immune response of the oral rabies vaccine SPBN GASGAS was compared to those elicited by a commercial inactivated vaccine using immunobridging. Based on the detection of rabies virus binding (rVBA) and rabies virus neutralizing antibodies (rVNA) as measured by ELISA and Rapid Fluorescent Focus Inhibition Test (RFFIT) the long-term immune response in dogs vaccinated orally with the SPBNA GASGAS strain of rabies vaccine in a bait was non-inferior to a conventional inactivated rabies vaccine. The outcome of this study supports extending the originally claimed duration of immunity (DOI) of SPBN GASGAS after oral vaccination for dogs from 6 to 30 months.

Evaluation of a new serological technique for detecting rabies virus antibodies following vaccination

Two major techniques are currently used to estimate rabies virus antibody values: neutralization assays, such as the rapid fluorescent focus inhibition test (RFFIT), and enzyme-linked immunosorbent assays (ELISAs). The RFFIT is considered the gold standard assay and has been used to assess the titer of rabies virus neutralizing antibodies for more than three decades. In the late 1970s, ELISA began to be used to estimate the level of rabies virus antibody and has recently been used by some laboratories as an alternate screening test for animal sera. Although the ELISA appears simpler, safer and more efficient, the assay is less sensitive in detecting low values of rabies virus neutralizing antibodies than neutralization tests. This study was designed to evaluate a new ELISA-based method for detecting rabies virus binding antibody. This new technique uses electro-chemi-luminescence labels and carbon electrode plates to detect binding events. In this comparative study, the RFFIT and the new ELISA-based technique were used to evaluate the level of rabies virus antibodies in human and animal serum samples. By using a conservative approximation of 0.15IU/ml as a cutoff point, the new ELISA-based technique demonstrated a sensitivity of 100% and a specificity of 95% for human samples and for experimental animal samples. The sensitivity and specificity for field animal samples was 96% and 95%, respectively. The preliminary results from this study appear promising and demonstrate a higher sensitivity than traditional ELISA methods.

Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

Characterization of protein involvement in rabies virus binding to BHK-21 cells

Characterization of protein involvement in rabies virus binding to BHK-21 cells

Characterization of protein involvement in rabies virus binding to BHK-21 cells.

Prior studies established the specificity of rabies virus receptors on BHK-21 cells based on the saturability of the receptors and on competitive binding. In the present study, we used protease-treated cells to identify the involvement of protein in the specific binding of rabies virus to these cells. In addition, biochemical characterization of n-octylglucoside solubilized BHK-21 plasma membranes demonstrated the involvement of a protease sensitive, heat insensitive, integral membrane protein or protein complex in rabies virus binding to these cells. The membrane component that binds rabies virus is associated with a high molecular weight fraction of the n-octylglucoside-plasma membrane extract isolated by gel filtration. This high molecular weight fraction (approximately 450 KDa) is enriched with a cell surface integral membrane component that comigrates with denatured bovine serum fibronectin (220 KDa). This cellular component did not bind polyclonal antisera to fibronectin in Western blot (native or denatured) or immunoprecipitation experiments. Direct and specific virus binding to high molecular weight plasma membrane protein(s) separated on Western blots further confirmed the role of a protein receptor in rabies virus binding to these cells.

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.

Rabies virus binding to an acetylcholine receptor α‐subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by α-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Membrane carbohydrate requirement for rabies virus binding to chicken embryo related cells.

The involvement of cellular carbohydrates in the interaction of rabies virus with chicken embryo related cell receptors was investigated. Two different methodological approaches were employed: specific glycosidases were used to treat the cells before infection, and specific lectins were used to compete with the viral binding. The results indicate the participation of sialic acid, galactose, mannose, and N-acetyl-glucosamine but not fucose in the cellular requirements for rabies virus infection.

Rabies virus binding to cellular membranes measured by enzyme immunoassay

The binding of rabies virus to cellular membranes was measured using an enzyme-linked immunosorbent assay (ELISA). Virus binding to membranes adsorbed to the wells of microtiter plates was detected with rabies virus antibody and alkaline phosphatase-linked second antibody. The greatest degree of binding was to myotube, neuroblastoma, and salivary gland membranes; intermediate levels occurred in striated muscle and nerve membranes; and low levels of binding were found in other membranes, including those of most parenchymal organs. Binding of rabies virus to myotube membranes was saturable, dependent on pH (with an optimum of pH 6.0), facilitated by the divalent cations Ca++, Mn++, and Mg++, and was temperature dependent. Binding was greatly reduced by inactivation of virus with beta-propiolactone or treatment of virus with trypsin. In embryonic chick myotubes, total acetylcholine receptor content and acetylcholinesterase activity undergo marked changes during development, first increasing and then decreasing at the time of hatching. Binding of rabies virus followed a similar pattern, indicating that the virus may interact with the acetylcholine receptor or other surface molecules undergoing similar developmental changes.

Rabies virus binding at neuromuscular junctions.

Morphological, immunocytochemical, biochemical, and immunological techniques have been used to describe rabies virus binding to a sub-cellular unit and molecular complex at the neuromuscular junction (NMJ). Early after infection in vivo, virus antigen and virus particles were found by immunofluorescence, electron microscopy and immunoelectron microscopy in regions of high density acetylcholine receptors (AChR) at NMJs. One monoclonal antibody (alpha-Mab) to the alpha subunit of the AChR blocked attachment of radio-labeled rabies virus to cultured muscle cells bearing high density patches of AChR. A sub-cellular structure, resembling an array of AChR monomers, bound both rabies virus antigens and alpha-Mab. By immunoblotting with electrophoretically transferred motor endplate proteins, rabies virus proteins and alpha-Mab bound to two proteins of 43 000 and 110 000 daltons. A rabies virus glycoprotein antibody detected virus antigen bound to the 110 000 dalton protein. An auto-immune (anti-idiotypic) response followed immunization of mice with rabies virus glycoprotein antigen; the antibody was directed to the 110 000 dalton protein. This auto-antibody altered the kinetics of neutralization by rabies virus antibody and induced the formation of rabies virus antibody after inoculation of mice. These results define, at the neuromuscular junction, a rabies virus receptor which may be part of the acetylcholine receptor complex.

Characterization of saturable binding sites for rabies virus.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin. Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum. Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus. Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes. Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell. Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus. Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses. Isolated rabies virus glycoprotein failed to compete in an equivalent manner. However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment. The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site. These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture.

Étude de l'adsorption et de la pénétration du virus rabique: Interactions avec les cellules BHK21 et des membranes artificielles

The binding of rabies virus and its entry into susceptible cells was studied using BHK21 cells and a liposomal membrane model of cellular constituents. When BHK21 cells were incubated with radiolabelled virus or purified glycoprotein, binding of viral material to cell surfaces was observed. Saturation of cellular membrane sites by unlabelled rabies glycoprotein did not inhibit binding of radiolabelled virus. This result suggested that the sites and/or the mechanism of binding of the glycoprotein molecules were independant, depending on whether glycoprotein was free in the incubating solution or anchored to the viral membrane. Binding of the virus to the cell membrane and entry of viral material were enhanced by incubating virus-cell mixtures in acidic conditions: the amount of virus radioactivity bound to cells was 10-fold higher at pH 5.4 than at pH 7.2, but the viral infectivity was the same. Furthermore, these two phenomenons required energy because lower amounts of viral radioactivity were detected when virus and cells were incubated at 4°C (instead of 20°C) with sodium azide. Attempts to saturate cell sites with unlabelled virus led to two types of results: (1) inhibition of binding of labelled virus by low concentrations. Rabies virus also bound to liposomes prepared with either cellular lipids alone or lipids and 0.01% Triton-X100 cellular extracts. This binding was specific only at pH 7.2 and only when virus was incubated with liposomes containing detergent extract. Under these conditions, liposomes could be partially saturated when incubated with unlabelled virus before contact with labelled virus. This observation suggests that virus bind to a detergent-soluble receptor. All these results led us to propose that the attachment of rabies virus to the BHK21-cell surface requires a specific receptor and that entry of viral material mainly involves a fusion of the viral membrane and the cell plasma membrane. L'attachement du virus rabique et sa pénétration dans une cellule permissive ont été étudiés à l'aide des modèles suivants: cellules BHK21 et membranes artificielles constituées d'éléments cellulaires. Lorsque des cellules BHK21 sont incubées en présence de virus rabique ou de glycoprotéine purifiée (préalablement marqués par incorporation de radioactivité) on enregistre un attachement très rapide du matériel radioactif sur la surface cellulaire. La saturation des sites cellulaires par la glycoprotéine non marquée n'inhibe pas l'attachement du virus marqué, suggérant que les sites et/ou le mécanisme d'attachement des molécules de glycoprotéine sont différents suivant que cette protéine est en solution ou solidaire de la particule virale. L'attachement du virus et son entrée dans les cellules sont augmentés lorsqu'on abaisse le pH de la solution d'incubation tout en conservant le pouvoir infectant du virus: la quantité de matériel viral radioactif lié aux cellules est 10 fois plus élevée à pH 5,4 qu'à pH 7,2. De plus, ces deux phénomènes nécessitent de l'énergie puisque des quantités plus faibles de radioactivité sont mises en évidence dans les cellules lorsque le virus marqué et les cellules sont incubés à 4°C (au lieu de 20°C) et en présence d'azoture de sodium. Lors des essais de saturation des sites cellulaires par le virus non marqué, on enregistre deux types de résultats pour l'attachement du virus marqué: inhibition pour les faibles concentrations de virus non marqué et activation pour les fortes concentrations. Le virus rabique se lie aussi aux liposomes préparés avec des lipides cellulaires, qu'on ait incorporé ou non à la préparation lipidique des constituants cellulaires solubles dans 0,01% de Triton-X100. Cette liaison n'est spécifique qu'à pH 7,2 et avec des liposomes contenant les produits cellulaires solubles dans le Triton-X100, seules conditions où le système peut être partiellement saturé. Cette observation suggère qu'un éventuel récepteur pour le virus rabique existe à la surface des cellules BHK et qu'il peut être extrait par un détergent. L'ensemble des résultats obtenus conduit à proposer que dans les présentes conditions expérimentales l'adsorption du virus rabique à la surface de la cellule BHK met en œuvre un récepteur spécifique et que la pénétration dans la cellule s'effectue majoritairement selon un mécanisme de fusion entre la membrane virale et la membrane plasmique cellulaire.