Rabies Virus Antigen Research Articles

Detection of rabies virus RNA in the central nervous system of experimentally infected mice using in situ hybridization with RNA probes

Rabies virus is usually demonstrated in human or animal tissues using antigen-detection or viral isolation techniques. Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (−) sense 35S- and 3H-labeled RNA probes, specific for rabies virus nucleocapsid protein mRNA, were used for the detection of rabies virus RNA in the nervous system of mice experimentally infected with fixed and street strains of rabies virus. In situ hybridization signals were compared with rabies virus antigen demonstrated with immunoperoxidase staining. Rabies virus RNA and antigen were-also demonstrated in the same neurons using a double-labeling technique. In situ hybridization has potential applications as a diagnostic test for rabies and in studies of rabies pathogenesis.

Rabies virus-specific T cell hybridomas: identification of class II MHC-restricted T-cell epitopes using synthetic peptides.

Rabies virus-specific T-cell hybridomas were produced from immune mice by somatic cell fusion. Cloned T-cell hybridomas were studied for antigen specificity using purified virus, a recombinant vaccinia virus expressing rabies glycoprotein and synthetic peptides containing amino acid sequences of rabies viral antigens. Two closely situated T-cell epitopes of rabies glycoprotein, and one of rabies nucleoprotein were identified using synthetic peptides corresponding to amino acid sequences of these proteins. The major histocompatibility gene complex elements that determine the recognition of antigen by these T-cell hybrids were determined using mouse fibroblasts (L cells) transfected with and expressing the I-Ad and I-Ed genes. Some of the T cell hybridomas exhibited significant cytotoxic activity against target cells expressing surface rabies antigens. This T cell mediated cytotoxicity requires cell-to-cell contact between target and effector cells since no by-stander cytotoxicity was observed. The results are discussed in the context of their significance for the design of newer subunit vaccines to prevent rabies infection.

Rabies Virus-specific Human T Cell Clones Provide Help for an in vitro Antibody Response against Neutralizing Antibody-inducing Determinants of the Viral Glycoprotein

Human T cell clones were prepared from peripheral blood mononuclear cells from a vaccinated human donor and kept in culture in the presence of rabies virus antigen and growth factors. Phenotypic analysis of the T cell clones revealed expression of the CD3 and CD4 cell surface markers, but not of CD8, consistent with a phenotype of helper/inducer T cells. The rabies virus specificity of the T cell clones was established by virus-specific proliferation in response to the rabies virus Pitman-Moore strain (PM) produced in three different cell substrates. The clones also responded to the rabies virus strains Evelyn-Rokitnicki-Abelseth (ERA) and challenge virus standard (CVS), but not to the rabies virus-related Mokola and Duvenhage-6 virus strains. Proliferative responses of T cell clones required rabies virus antigen to be presented by autologous antigen-presenting cells in association with HLA class II molecules. When cultured with rabies virus antigen, but in the absence of growth factors, some of the T cell clones provided help for an antibody response of rabies virus immune B lymphocytes. Analysis of culture supernatant fluids showed that at least a part of this antibody response was directed against neutralizing antibody-inducing determinants of the viral glycoprotein.

Enhancement of antigen-specific interleukin 2 production by adding liposomes to rabies antigens for priming

Antigen-specific IL-2 production was assessed, using splenocytes from rabies immune mice incubated for 24 h with rabies virus antigen. The antigenic material used for in vivo priming was either purified glycoprotein from rabies virus, or the inactivated virus. The time between priming, harvesting and restimulation of the splenocytes was 7 days. It was found that when antigenically inert liposomes were injected, together with antigenic material, to the prospective splenocyte donor mice, IL-2 production was enhanced. This augmentation was observed particularly when priming was performed with the inactivated rabies virus.

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.

Immunohistochemical staining of rabies virus antigen with monoclonal and polyclonal antibodies in paraffin tissue sections.

SummaryImmunohistochemical staining methods (indirect immunoperoxidase, avidin‐biotin‐complex) with monoclonal and polyclonal antibodies were used to demonstrate rabies ribonucleoprotein (RNP) in paraffin sections of formalin‐fixed brain tissue of 28 naturally infected wild and domestic animals. Especially with the monoclonal antibody the staining contrast was very high. Obviously, this monoclonal antibody tested among others detects an epitope on the N protein which is resistant to the conventional fixation and embedding procedure. In all 28 cases virus infection was found to extend to the brain regions with a relative predilection for the hippocampal formation, the diencephalon, and the brainstem. Virus antigen was mainly present in the nerve cell perikarya and processes but was also found in glial cells, especially in the interfascicular oligodendrocytes, suggesting the existence of a broader cell tropism of rabies virus in the natural course of infection. Moreover, two cases of non‐rabid animals (sheep, elk) with pseudo‐Negri bodies in the nerve cells of the Ammon's horn showed a negative staining result. The immunohistochemical method described facilitates the diagnosis or the exclusion of rabies in diagnostic pathology, especially if only formalin‐fixed brain tissue is available.ZusammenfassungImmunhistochemische Färbung von Tollwutvirus‐Antigen mit monoklonalen und polyklonalen Antikörpern in Paraffin eingebetteten GewebeschnittenImmunhistochemische Färbemethoden (indirekte Immunperoxidase, Avidin‐Biotin‐Komplex) mit mono‐ und polyklonalen Antikörpern wurden angewandt, um Rabies Ribonukleoprotein (RNP) in Paraffinschnitten formalinfixierten Hirngewebes darzustellen. Untersucht wurden 28 natürlich infizierte Wild‐ und Haustiere. Speziell mit dem monoklonalen Antikörper erhält man sehr kontrastreiche Darstellungen. Offensichtlich erfaßt dieser unter mehreren gestesteten monoklonalen Antikörpern ein Epitop des N‐Proteins, welches die Fixierung und den Einbettungsprozeß unbeschadet übersteht. Die Virusinfektion fand sich in allen Hirnregionen der 28 Tiere ausgebreitet, mit einem Schwerpunkt in der Hippocampusformation, dem Zwischenhirn und dem Hirnstamm. Neben den hauptsächlich befallenen Nervenzellen war Virusantigen aber auch in der Glia, vor allem den interfaszikulären Oligodendrozyten, nachweisbar, was darauf hinweist, daß das Wirtszellspektrum bei natürlich verlaufender Tollwut größer als bisher vermutet ist. Darüber hinaus wurden zwei Tiere (Schaf, Elch) ohne Tollwut, jedoch mit Pseudo‐Negrikörperchen in den Nervenzellen des Ammonshornes untersucht, wobei diese intrazytoplasmatischen Einschlüsse immunhistochemisch nicht reagierten. Die vorgestellte immunhistochemische Methode ermöglicht die Diagnose oder den Ausschluß einer Tollwut in der diagnostischen Pathologie, vor allem dann, wenn nur noch formalinfixiertes Hirngewebe zur Verfügung steht.

The association of the rabies glycoprotein with liposome (immunosome) induces an in vitro specific release of interleukin 2

BALB/c mice were primed by receiving a unique intraperitoneal injection of rabies virus antigens presented as complete inactivated virus (P.V. strain) or as purified glycoproteins either in the aggregated form or in physical combination with liposomes (i.e., in the form of “immunosomes”). The splenocytes of these mice were restimulated, 6–15 days after priming, in culture with rabies virus antigens, and antigen-specific IL-2 production was measured. It was found that rabies antigens presented as immunosomes were as active as the inactivated virus, whereas equivalent amounts of purified glycoproteins were inactive. The optimal amounts of rabies immunosomes used for priming was found to be 0.5 to 0.05 μg per mouse.

Electrophysiological and sleep alterations in experimental mouse rabies

Changes in the spontaneous brain electrical activity and sleep organization were investigated in 5 mice strains during the evolution of experimental fixed rabies infection. Cortical electrodes were chronically implanted for continuous EEG recording and spectral analysis until death. Three evolutionary phases were individualized. The initial phase exhibited alterations of sleep stages, REM sleep disappearance, pseudoperiodic facial myoclonus and first clinical signs. The mature phase was characterized by a generalized EEG slowing (2–4 cycles/s). The terminal phase occurring with extinction of hippocampal rhythmic slow acticity showed a flattening of cortical activity. The brain electrical activity ceased about 30 min before the cardiac arrest. Paroxysmal activities appeared during the course of the disease as burst of rhythmic slow waves, pseudoperiodic spikes or occasionally ictal epileptic discharges. Spectral analysis revealed a progressive and characteristic clustering of the EEG frequency band power values. The spread of infection in the CNS was monitored by specific immunofluorescence studies which revealed the presence of rabies virus antigen in the pons, the cerebellum, the thalamus and the cortex during the initial phase. The pyramidal field of the hippocampus was infected during the mature phase but the gyrus dentatus was never infected even at the terminal phase. These studies show that particular neuronal functions are impaired during rabies virus infection suggesting that neuronal alterations may be involved in the pathogenic mechanisms leading to lethality.

Use of immunofluorescence examination to detect rabies virus antigen in the skin of humans with clinical encephalitis.

Use of immunofluorescence examination to detect rabies virus antigen in the skin of humans with clinical encephalitis.

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.

Immunoelectron microscopic localization of rabies virus antigen in central nervous system and peripheral tissue using low-temperature embedding and protein A-gold

A protein A-gold technique was used in conjunction with low temperature embedding to visualize ultrastructurally mature virions and sites of viral replication in the brains of rabies virus-infected mice after peripheral inoculation of virus. The association of viral profiles and gold particles with synaptic membranes, microtubules and rough endoplasmic reticulum suggested a mechanism of rabies virus transport within the central nervous system. Early interactions of inflammatory cells with the virus inoculum were characterized by phagocytosis by non-degranulating neutrophils and mononuclear cells.

Inhibition of Rous sarcoma virus-induced transformation by preinfection with rhabdoviruses.

In vivo preinfection of chicks with rabies virus (RV) or vesicular stomatitis virus (VSV) ts 1026 inhibits tumour formation after superinfection with Rous sarcoma virus (RSV). The degree of inhibition depends on the titre of the infecting viruses and the interval between rhabdovirus and RSV infection. In vitro, cells preinfected with VSV ts 1026 under non-permissive conditions and superinfected with RSV, are not transformed as judged by cell morphology, serum requirement for growth or the capacity to form colonies in soft agar, all these being the same as in uninfected cells. Doubly infected cells take up less deoxyglucose than cells infected with RSV only and more than cells infected with VSV only. RSV multiplication in inhibited in doubly infected cells: the supernatant fluid of these cells contains fewer focus-forming units and less reverse transcriptase activity than that of cells infected with RSV only. Doubly infected cells contain both VSV and RSV internal antigens 15 days after infection. The supernatant fluid of cells infected with VSV and maintained under non-permissive conditions inhibits transformation by RSV and multiplication of RSV, but not of VSV. Under non-permissive conditions, the rhabdoviruses undergo at least part of the infectious cycle, but no infectious virus is produced. RV antigen can be detected in the brain of parenterally infected chicks and VSV antigen in cells infected 15 days previously. We conclude that the inhibition of RSV multiplication and expression is probably due to one or more processes linked to the persistence of rhabdovirus components and that it cannot be attributed exclusively to interferon.

Safety tests with Flury HEP strain 675 in wild-living European mammals

For the evaluation of residual pathogenicity of Flury HEP strain 675, the vaccinal virus was orally administered to 207 animals belonging to 15 wild-living and 1 domestic species. Rabies virus antigen could be demonstrated by FA method in 20 animals. Reisolation of the virus was possible from 23 animals within the first two weeks after application, the titres being very low. During the observation period of 100 days no symptoms of rabies could be observed in the animals tested.

Purification, concentration and evaluation of rabies virus antigen

A number of concentration and purification procedures for rabies virus antigen have been developed during the last decades but only ultrafiltration and density gradient centrifugation have been found suitable for large scale production. The antibody binding test developed by Arko, Wiktor and Sikes was modified and was found useful for evaluation of inactivated antigenconcentrates. This test was used as an ‘in process’ control. Three examples of practical experience are reported and discussed.

Entry of Rabies Virus into the Peripheral Nerves of Mice

Young adult mice were inoculated in the hind limb with rabies virus or Sindbis virus. Rabies 1820B virus antigen was detected in leg sections by immuno-fluorescence at 1 h post-inoculation at sites comparable in form and distribution to cholinesterase-positive sites, which represent motor end-plates (MEPs). Sites which were rabies virus antigen-positive by immunofluorescence were also cholinesterase- positive on double-stained slides. Rabies CVS virus detected by autoradiography was similarly distributed at 6 h post-inoculation. Uptake of rabies virus at motor nerve endings was confirmed by the detection of rabies antigen by immunofluorescence in ventral horn cells in the spinal cord at 20 h post-inoculation before involvement of dorsal root ganglia. Rabies virus antigen could not be detected at MEPs if the virus had been inactivated by beta propiolactone or mixed with antibody prior to injection or if the sciatic nerve had been cut 7 days earlier, similarly treated groups of mice survived for the observation period of 6 weeks. Rabies virus antigen was found at MEPs in mice given antibody 24 h before virus injection, but virus antigen was not found in the spinal cord, and mice similarly treated survived. Sindbis virus strain Ar86, which like rabies virus is neurotropic in adult mice, was also found at MEPs and in peripheral nerves by autoradiography at 6 h post-inoculation. In contrast to results with rabies virus-infected mice, stimulation of the sciatic nerve for the first hour post-inoculation prevented mortality. Sindbis virus strain Ar339, which is not neurotropic in adult mice, could not be detected at MEP's by immunofluorescence or autoradiography and mice injected with virus survived. The results presented here suggest that rabies virus and perhaps other neurotropic viruses can use the motor axon terminal at the neuromuscular junction as a site of entry into the nervous system.

Influence of cell type and virus upon lysis of rabies virus-infected cells by antibody and complement.

Mouse neuroblastoma (MNB) cells infected with ERA virus were specifically lysed in the presence of rabbit complement by antisera produced in mice to challenge virus standard (CVS), ERA, Flury HEP or street virus (SV) strains of rabies. MNB or EL-4 cells persistently infected with ERA, MNB cells infected with CVS, and BHK-21/S13 cells infected with ERA or Flury HEP also were suitable targets. CER cells infected with either ERA, CVS or Flury HEP, BHK-21/S13 cells infected with CVS and MNB cells infected with Flury HEP were not suitable targets. Two unusual findings indicated that 1. some cells which were greater than 80 percent positive for rabies viral membrane antigen(s) were poorly lysed, and 2. some cells that expressed cytoplasmic antigen lacked detectable membrane antigens.

The antibody-binding-test, a useful method for quantitative determination of inactivated rabies virus antigen

The antibody binding test after some modifications was found useful for quantitative determination of rabies virus antigen. Reproducibility studies showed that results of repeated tests were less variable when compared with the NIH test. The advantages of the ABT are discussed. This test seems to be more exact, more reliable and more reproducible than the NIH test and is a good method for in process control of inactivated Rabies virus antigen.

Identification of rabies virus antigen in the skin of foxes.

Identification of rabies virus antigen in the skin of foxes.

Immune responses of humans to a human diploid cell strain of rabies virus vaccine: lymphocyte transformation, production of virus-neutralizing antibody, and induction of interferon.

Lymphocyte transformation, production of neutralizing antibody, and interferon activity of 20 healthy volunteers in response to a human diploid cell strain (HDCS) of rabies virus vaccine were studied. Ten vaccines received 1.0 ml of HDCS vaccine on days 0, 3, 7, and 14, and five of these volunteers received, in addition, 20 international units of human rabies immune globulin/kg of body weight on day 0. All 10 volunteers developed high titers of neutralizing antibody, and eight of the 10 had lymphocytes that were immunologically stimulated by HDCS rabies virus antigen. The interferon responses of eight vaccines to 1.0 ml of HDCS vaccine given intramuscularly on days 0 and 28 and of two vaccines to 1.0 ml of vaccine given intradermally in eight body sites on the same days were measured; low levels of interferon-like activity were found in eight of nine volunteers after the first vaccination, and no activity was found upon revaccination. The high titers of neutralizing antibody that developed did not correlate with lymphocyte stimulation or interferon-like activity, but it is true that all 20 volunteers did develop high titers of neutralizing antibody after 1.0 ml of HDCS vaccine was given intradermally.

Kinetics of different specific immunological parameters after rabies vaccination in mice.

A significant protection to an intracerebral challenge of 70 mean lethal doses of a standard live rabies virus strain was obtained in BCG-pretreated mice or in normal mice which had been immunized with a single subcutaneous injection of a beta-propiolactone-inactivated rabies vaccine. Concomitantly, levels of delayed-type hypersensitivity (measured in vivo by the footpad test) and serum-neutralizing activity were evaluated at various times after immunization. All immune criteria were significantly augmented in the BCG-pretreated, rabies-immune mice as compared to normal, rabies-immune mice. However, peak levels of protection, delayed-type hypersensitivity, and serum-neutralizing activity did not occur at the same times. For instance, in the BCG-pretreated, rabies-immune mice, delayed-type hypersensitivity peaked on day 7, protection peaked on day 21, and serum-neutralizing activity peaked on day 60. In BCG-pretreated mice, which did not receive the rabies vaccine, positive delayed-type hypersensitivity, some protection, and serum neutralizing activity were observed 4 to 5 weeks after BCG pretreatment. The possible relationships between specific and nonspecific immunity provoked by rabies virus antigens, tissue culture cell-associated antigens (derived from the bovine fetal kidney cells in which the rabies virus was grown, and BCG are discussed.