RESUMEN
Intracellular NP oligomers have been shown to react with some anti-NP monoclonal antibodies (mAbs) in radio-immnoprecipitation, immunoblotting, and dot immunoassay. Soluble NP monomers obtained after thermal dissociation of NP oligomers are not recognized by mAbs unlike the NP monomers whose concentration increased by about 100-fold due to transfer to the nitrocellulose membrane after polyacrylamide gel electrophoresis. The findings demonstrated that in the intact NP oligomers there were epitopes determined by their quaternary structure. These oligomer-dependent epitopes may be renaturated in vitro under the conditions allowing for a concentration-dependent NP-NP association.
Asunto(s)
Epítopos/inmunología , Subtipo H3N8 del Virus de la Influenza A/inmunología , Proteínas de la Nucleocápside/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Línea Celular , Perros , Mapeo Epitopo , Epítopos/metabolismo , Calor , Immunoblotting , Proteínas de la Nucleocápside/metabolismo , Conformación Proteica , Ensayo de Radioinmunoprecipitación , SolubilidadRESUMEN
Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.
Asunto(s)
Acetilcolina/metabolismo , Enfermedad de Borna/metabolismo , Bornaviridae/patogenicidad , Corteza Cerebral/metabolismo , Fibras Colinérgicas/metabolismo , Encefalitis Viral/metabolismo , Proteínas de Transporte de Membrana , Infecciones por Mononegavirales/metabolismo , Proteínas de Transporte Vesicular , Acetilcolinesterasa/metabolismo , Animales , Enfermedad de Borna/patología , Enfermedad de Borna/fisiopatología , Proteínas Portadoras/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/virología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/patología , Fibras Colinérgicas/virología , Progresión de la Enfermedad , Regulación hacia Abajo/inmunología , Encefalitis Viral/patología , Encefalitis Viral/fisiopatología , Inmunohistoquímica , Infecciones por Mononegavirales/patología , Infecciones por Mononegavirales/fisiopatología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/virología , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Prosencéfalo/metabolismo , Prosencéfalo/patología , Prosencéfalo/virología , Ratas , Ratas Endogámicas Lew , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8(+) T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426-434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (10(6) FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8(+) T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.
Asunto(s)
Enfermedad de Borna/inmunología , Nucleoproteínas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Femenino , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.
Asunto(s)
Virus de la Enfermedad de Borna/química , Linfocitos T CD8-positivos/química , Antígenos de Histocompatibilidad Clase I/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Epítopos/química , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8(+) T cells are responsible for the destruction of virus-infected brain cells. CD4(+) T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Enfermedad de Borna/prevención & control , Virus de la Enfermedad de Borna/inmunología , Encefalitis Viral/prevención & control , Glicoproteínas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/sangre , Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/aislamiento & purificación , Virus de la Enfermedad de Borna/patogenicidad , Línea Celular , Enfermedad Crónica , Encefalitis Viral/inmunología , Femenino , Inmunohistoquímica , Masculino , Pruebas de Neutralización , Ratas , Ratas Endogámicas Lew , Proteínas Virales/inmunologíaRESUMEN
Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Granulocitos/virología , Trastornos Mentales/virología , Infecciones por Virus ARN/virología , Proteínas Virales/genética , Virus de la Enfermedad de Borna/aislamiento & purificación , Virus de la Enfermedad de Borna/patogenicidad , Fraccionamiento Celular , Granulocitos/inmunología , Humanos , Trastornos Mentales/inmunología , ARN Viral/análisis , Análisis de Secuencia de ARNRESUMEN
Experimental infection of rats with Borna disease virus (BDV) and natural BDV infection of horses and sheep leads to a virus-induced T-cell-mediated immunopathology in the central nervous system. Earlier work revealed the importance of the BDV-specific T-cell response and of CD8(+) effector cells in particular in the destruction of virus-infected cells. Evidence was also presented that this major histocompatibility complex class I-restricted lysis detected in vitro might play a functional role in the immunopathogenesis of Borna disease. The present study employed different vaccinia virus recombinants expressing single BDV-specific proteins to investigate the specificity of the cytolytic CD8(+)-T-cell response, revealing a major epitope on the BDV nucleoprotein p40. In contrast, no direct evidence in favor of the presence of in vivo relevant cytotoxic T-lymphocyte epitopes on other BDV-specific proteins was found.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Enfermedad de Borna/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Virales/inmunología , Animales , Línea Celular , Cricetinae , Femenino , Ratas , Ratas Endogámicas LewRESUMEN
Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Enfermedad de Borna/inmunología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/patogenicidad , Animales , Animales Recién Nacidos , Antígenos Virales/metabolismo , Sistema Nervioso Autónomo/virología , Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/aislamiento & purificación , Sistema Nervioso Central/virología , Femenino , Inmunización Pasiva , Inmunocompetencia , Técnicas In Vitro , Masculino , Pruebas de Neutralización , Especificidad de Órganos , Nervios Periféricos/virología , Ratas , Ratas Endogámicas Lew , Factores de TiempoAsunto(s)
Virus de la Enfermedad de Borna/aislamiento & purificación , ARN Viral/aislamiento & purificación , Esquizofrenia/virología , Trastornos Somatomorfos/virología , Virus de la Enfermedad de Borna/genética , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificaciónRESUMEN
The antiviral effect of amantadine (1-aminoadamantane) was tested in vitro as well as in vivo. Treatment of persistently Borna disease virus (BDV)-infected cell lines of different origin and for various length of time did not result in a general reduction of virus titer or clearance of virus from infected cells. In vivo, rats were treated with amantadine by daily oral application or by use of osmotic pumps, and in both cases treatment was started before infection. Neither route of application of the drug had any influence on the time of onset of disease, on antiviral antibody titers, on virus titer in the brain, on the severity of the inflammatory reaction in the brain, or on the severity of neurological symptoms. These experiments, although revealing negative results and obtained using a virus from a natural case of Borna disease grown after isolation in vitro for a long period of time, should caution from the general use of amantadine as a curative agent against BDV infection as has been implicated recently [Bode et al. (1997) Lancet 349:178-179].
Asunto(s)
Amantadina/farmacología , Antivirales/farmacología , Enfermedad de Borna/tratamiento farmacológico , Virus de la Enfermedad de Borna/efectos de los fármacos , Administración Oral , Amantadina/sangre , Amantadina/uso terapéutico , Animales , Antivirales/uso terapéutico , Enfermedad de Borna/patología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/fisiología , Encéfalo/patología , Línea Celular , Perros , Encefalitis Viral/tratamiento farmacológico , Encefalitis Viral/patología , Encefalitis Viral/virología , Femenino , Cobayas , Humanos , Ratas , Ratas Endogámicas Lew , Replicación Viral/efectos de los fármacosRESUMEN
Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that persistently infects warm-blooded animals. In horses and other natural animal hosts, infections with BDV cause meningoencephalitis and behavioral disturbances. Experimental infection of adult mice takes a nonsymptomatic course, an observation previously believed to indicate that this animal species is not suitable for pathogenesis studies. We now demonstrate that BDV frequently induces severe neurological disease in infected newborn mice. Signs of neurological disease were first observed 4 to 6 weeks after intracerebral infection. They included a characteristic nonphysiological position of the hind limbs at an early stage of the disease and paraparesis at a later stage. Histological examination revealed large numbers of perivascular and meningeal inflammatory cells in brains of diseased mice and, unexpectedly, no increase in immunoreactivity to glial fibrillar acidic protein. The incidence and severity of BDV-induced disease varied dramatically among mouse strains. While only 13% of the infected C57BL/6 mice showed disease symptoms, which were mostly transient, more than 80% of the infected MRL mice developed severe neurological disorder. In spite of these differences in susceptibility to disease, BDV replicated to comparable levels in the brains of mice of the various strains used. Intracerebral infections of newborn beta2-microglobulin-deficient C57BL/6 and MRL mice, which both lack CD8+ T cells, did not result in meningoencephalitis or neurological disease, indicating that the BDV-induced neurological disorder in mice is a cytotoxic T-cell-mediated immunopathological process. With this new animal model it should now be possible to characterize the disease-inducing immune response to BDV in more detail.
Asunto(s)
Enfermedad de Borna/inmunología , Meningoencefalitis/virología , Animales , Animales Recién Nacidos , Enfermedad de Borna/fisiopatología , Enfermedad de Borna/virología , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Proteína Ácida Fibrilar de la Glía/inmunología , Cinética , Meningoencefalitis/inmunología , Ratones , Ratones Endogámicos , Ratas , Especificidad de la Especie , Replicación ViralRESUMEN
Persistent Borna disease virus infection of the brain can be prevented by treatment of naive rats with a virus-specific CD4+ T-cell line prior to infection. In rats receiving this treatment, only a transient low-level encephalitis was seen compared to an increasingly inflammatory reaction in untreated infected control rats. Virus replication was found in the brain for several days after infection before the virus was cleared from the central nervous system. The loss of infectivity from the brain was confirmed by negative results by reverse transcription-PCR with primers for mRNA, by in situ hybridization for both genomic and mRNA, and by immunohistology. Most importantly, in vitro assays revealed that the T-cell line used for transfusion had no cytotoxic capacity. The kinetics of virus clearance were paralleled by the appearance of CD8+ T cells and the expression of perforin in the brain. Testing of lymphocytes isolated from the brains of CD4+ T-cell-treated rats after challenge revealed high cytotoxic activity due to the presence of CD8+ cytotoxic T cells at time points when brain lymphocytes from infected control rats induced low-level cytolysis of target cells. Neutralizing antiviral antibodies and gamma interferon were shown not to be involved in the elimination of virus from the brain.
Asunto(s)
Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/inmunología , Encéfalo/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Antivirales/inmunología , Enfermedad de Borna/virología , Encéfalo/virología , Linfocitos T CD4-Positivos/virología , Línea Celular , Trasplante de Células , Femenino , Glicoproteínas de Membrana/metabolismo , Pruebas de Neutralización , Perforina , Proteínas Citotóxicas Formadoras de Poros , Ratas , Ratas Endogámicas Lew , VacunaciónRESUMEN
Rats experimentally infected with the highly neurotropic Borna disease virus (BDV) display a wide variety of dysfunction such as learning deficiencies and behavioral abnormalities. Prior to the onset of encephalitis alterations of one of the major cortical neurotransmitters, acetylcholine, were monitored immunohistochemically by light and electron microscopy of its synthesizing enzyme choline acetyltransferase (ChAT). We found a progressing decrease in the number of ChAT-positive fibers, starting with discrete changes at day 6 post infection (p.i.) and ending with a nearly complete loss of cholinergic fibers, especially in the hippocampus and neocortex, suggesting a massive disturbance of the cholinergic innervation by day 15 p.i.. The fiber pathways (e.g., fimbria-fornix) connecting the basal forebrain with these target areas in the cortex displayed axon spheroids which are often linked to axonal transport dysfunction. No evidence for significant cellular destruction was seen in the brain, including the cells of origin of these axons in the basal forebrain. We conclude that the motor, mood, learning and memory disabilities in BDV-infected rats are likely to result, in part, from cortical cholinergic denervation. The present study gives new insights into the pathogenesis of neurological disease caused by a noncytopathogenic virus.
Asunto(s)
Acetilcolina/fisiología , Enfermedad de Borna/patología , Corteza Cerebral/patología , Encefalitis/patología , Animales , Anticuerpos Monoclonales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Colina O-Acetiltransferasa/análisis , Hipocampo/patología , Ratas , Ratas Endogámicas LewAsunto(s)
Enfermedad de Borna/epidemiología , Virus de la Enfermedad de Borna/aislamiento & purificación , Trastornos Psicóticos/virología , ARN Viral/sangre , Animales , Enfermedad de Borna/diagnóstico , Alemania/epidemiología , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Ratas , Estudios SeroepidemiológicosRESUMEN
Lymphocytes were isolated from the brains of Borna disease virus-infected donor Lewis rats at various time points after infection. Cell populations were characterized by cytofluorometry, with special emphasis on CD4+ and CD8+ cells. Testing of isolated lymphocytes revealed major histocompatibility complex class I-restricted cytotoxic activity. Reverse transcription-PCR analyses of brain homogenates of infected donors revealed the presence of CD8 mRNA after day 11 of infection and of perforin mRNA between days 13 and 25 after infection. Adoptive transfers of lymphocytes isolated from the brain at days 13 and 21 resulted in severe neurological symptoms, resembling experimental Borna disease. The onset of disease was dependent on the cell numbers transferred and was clearly related to the appearance of T cells in the brain. CD8+ T cells were found in the parenchyma, whereas CD4+ T cells were found predominantly in perivascular locations. A disseminated lymphocytic infiltration in the parenchyma was accompanied by severe morphological alterations, including significant necrosis of neurons. Furthermore, a prominent spongiform-like degeneration was observed; this increased over time and finally resulted in severe cortical brain atrophy. Lymphocytes obtained during the beginning chronic phase of experimental Borna disease in rats had no significant cytolytic capacity in vitro and were also not able to induce neurological symptoms typical of Borna disease after adoptive transfer. The data presented here show for the first time that lymphocytes isolated from the site of the inflammatory lesions, namely, the brains of diseased rats, induce the immunopathological reaction and cause Borna disease. After transfer, the pathological alterations induced in the recipients exactly reflect those observed during experimentally induced Borna disease in rats, including necrosis of neurons and glial cells and gross degeneration resulting in cortical brain atrophy. Evidence that the immunopathology of Borna disease is closely related to the presence of CD8+ T cells in the brain parenchyma is provided.
Asunto(s)
Traslado Adoptivo , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/patogenicidad , Encéfalo/virología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Glicoproteínas de Membrana/inmunología , Animales , Enfermedad de Borna/inmunología , Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/inmunología , Encéfalo/citología , Encéfalo/inmunología , Encéfalo/patología , Cuello del Útero/virología , Encefalitis Viral , Femenino , Ganglios Linfáticos/virología , Glicoproteínas de Membrana/genética , Degeneración Nerviosa , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero , Conejos , Ratas , Ratas Endogámicas LewRESUMEN
Tissues from nine horses and one donkey suffering from natural Borna disease were investigated. Clinically, all animals demonstrated progressive reduced mentation and aggravating gait disturbances. During the clinical course anorexia and progressive loss of proprioception were observed. Cranial nerve failure was accompanied by signs of pharyngeal paralysis, sialorrhea, bruxism, and by blindness. Virologically, infectious virus was detected in the brain of all animals investigated but was not found regularly in all areas of the brain. However, in all cases, infectivity was found in the thalamus and the hippocampal area. In contrast, in other compartments of the central nervous system virus was not detected regularly. These findings correlate well to the results obtained from assaying Borna virus-specific RNA by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and virus specific antigen by immunohistochemistry. No evidence of infection was found in the autonomic nervous system and peripheral organs. Immunomorphologically, lymphocytic inflammatory reactions and increased expressions of MHC class I and class II antigen were found in the brain as well as in the trigeminal and olfactory system. No evidence of inflammation was found in the retina. From the finding that BDV-proteins and nucleic acids were even more abundant in the trigeminal system as compared to the olfactory system, we conclude that infection may have occurred via the trigeminal nerve.
Asunto(s)
Enfermedad de Borna , Enfermedades de los Caballos , Animales , Conducta Animal , Enfermedad de Borna/inmunología , Enfermedad de Borna/patología , Enfermedad de Borna/fisiopatología , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/aislamiento & purificación , Encéfalo/inmunología , Encéfalo/virología , Equidae , Femenino , Enfermedades de los Caballos/inmunología , Enfermedades de los Caballos/patología , Enfermedades de los Caballos/fisiopatología , Enfermedades de los Caballos/virología , Caballos , Masculino , Sistema Nervioso/patología , Sistema Nervioso/fisiopatología , Sistema Nervioso/virología , Proteínas Virales/análisisRESUMEN
In order to replace the radioactive 51chromium release assay (CRA), a colorimetric method for the determination of cell-mediated cytotoxicity of natural killer (NK) effector cells of dogs and adherent target cells was developed using the dye Rose Bengal (RB). After a 14 h incubation period of leucocytes isolated from the peripheral blood (PBL) of dogs and a natural killer cell-sensitive canine adenocarcinoma cell line (CTAC), effector and lysed target cells were removed by washing, and the surviving adherent target cells were stained with RB. The optical density (OD) of the remaining target cells was measured in a microspectrophotometer (ELISA reader) and was found to correspond to the number of surviving cells, and thus was inversely correlated to the cytotoxic activity. The RB assay revealed almost identical cytotoxic values when compared with the CRA. In contrast to this assay the RBA is quick and easy to perform, inexpensive and avoids radioactive materials and waste. However, the method is restricted to adherent target cells.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/veterinaria , Adenocarcinoma/veterinaria , Animales , Adhesión Celular/inmunología , Línea Celular , Células Cultivadas , Radioisótopos de Cromo , Colorimetría/veterinaria , Perros , Inmunidad Celular , Células Asesinas Naturales/inmunología , Cinética , Recuento de Linfocitos , Rosa Bengala , Coloración y Etiquetado , Neoplasias de la Tiroides/veterinariaRESUMEN
Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Enfermedad de Borna/inmunología , Mapeo Epitopo , Fosfoproteínas/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Enfermedad de Borna/sangre , Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/genética , Línea Celular , Reacciones Cruzadas , Estructura Molecular , Fosfoproteínas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Virales/genéticaRESUMEN
A variety of viruses can cause diseases of the central nervous system by two different mechanisms. Cytolytic viruses exert direct effects on cells of the nervous system that result in death and cell loss. Noncytolytic viruses do not directly damage cells, but might harm cell functions in the long run. However, more common is the reaction of the immune system towards viral antigens of those noncytolytic viruses which ultimately might lead to an immunopathological disease. Several peculiarities of the central nervous system with regard to an immune reaction within this organ are discussed in the context of viral infections.