RESUMEN
We demonstrated the experimental vertical transmission of Borna disease virus (BDV) in pregnant BALB/c mice. Giessen strain He/80 of BDV was used in the present study. Six six-week-old mice were inoculated intraperitoneally with 10(5) 50% tissue culture infective doses (TCID50), and were bred immediately. Four pregnant mice were sacrificed under anaesthesia on the 10th and 14th days after vaginal plug formation. Nine newborns from two maternal mice were sacrificed under anaesthesia on the 7th day after birth. Positive signals with RT-nested PCR techniques for BDV p24-RNAs were seen in the fetuses, placentas and brains of all newborn mice. No immunopositivities for BDV p40 were found in the fetuses or placentas at 10 days' gestation. BDV p40 immunopositivities were found in neurons of the fetal brains and in decidual cells of the placentas at 14 days' gestation. They were also found in neurons of the brains of newborn mice. At 10 days' gestation, no positive signals for BDV p40 sense or antisense riboprobes were seen in the fetal brains or placentas. Positive signals were found in neurons of the fetal brains and decidual cells of the placentas at 14 days' gestation. Positive signals for BDV p40 sense and antisense riboprobes were found in almost all neurons throughout the brains of nine newborn mice. These results suggest that persistent infection with BDV in newborn mice may be induced by vertical transmission during gestation.
Asunto(s)
Enfermedad de Borna/transmisión , Virus de la Enfermedad de Borna/aislamiento & purificación , Modelos Animales de Enfermedad , Transmisión Vertical de Enfermedad Infecciosa , Animales , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/genética , Encéfalo/virología , Femenino , Feto/virología , Humanos , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/virología , ARN Viral/análisis , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Borna disease (BD) was diagnosed in a 3-year-old male Welsh corgi suffering from a severe and acute progressive disorder of the central nervous system. Histopathologically, neuronal lesions were characterized by a non-suppurative encephalomyelitis dominated by large perivascular cuffs consisting of lymphocytes, macrophages and plasma cells; also present were inflammatory cell infiltrates in the neural parenchyma, neuronophagia and focal gliosis. Strong immunolabelling with BD virus (BDV) p40 antibody was diffusely distributed in the cytoplasm of small and large neurons in areas of the brain with and without inflammatory changes, and also in the spinal cord. Positive hybridization signals with BDV p40 sense and antisense riboprobes were seen in the nucleus and cytoplasm of the neurons throughout the whole brain and spinal cord. BDV p24 RNA in formalin-fixed brain tissue was detected by reverse transcriptase (RT)-nested polymerase chain reaction (PCR). BDV p24 RNA-positive signals were detected in the temporal lobe. This is the first report of spontaneous canine BD in Japan.
Asunto(s)
Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/aislamiento & purificación , Sistema Nervioso Central/patología , Enfermedades de los Perros/patología , Animales , Antígenos Virales/análisis , Enfermedad de Borna/metabolismo , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/virología , Enfermedades de los Perros/metabolismo , Perros , Hibridación in Situ/veterinaria , Japón , Masculino , Neuronas/metabolismo , Neuronas/patología , Reacción en Cadena de la Polimerasa/veterinaria , ARN Viral/análisis , Proteínas Virales/análisis , Proteínas Virales/genética , Proteínas Virales/metabolismoAsunto(s)
Enfermedad de Borna/patología , Virus de la Enfermedad de Borna/patogenicidad , Enfermedades de los Bovinos/virología , Enfermedad Aguda , Animales , Antígenos Virales/análisis , Virus de la Enfermedad de Borna/genética , Bovinos , Progresión de la Enfermedad , Femenino , Inmunohistoquímica , Japón , ARN Viral/análisisRESUMEN
The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.
Asunto(s)
Virus de la Enfermedad de Borna/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Células COS , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Perros , Proteína HMGB1 , Humanos , Líquido Intracelular/metabolismo , Neuritas/fisiología , Neuronas/citología , Neuronas/metabolismo , Fosfoproteínas/genética , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/genéticaRESUMEN
Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that causes a chronic neurological disease in a wide variety of animal species. To develop a better understanding of the correlation between neurological disorders caused by BDV infection and virus distribution in the brain, we investigated viral dynamics in the central nervous system (CNS) of neonatally BDV-infected gerbils during the late stage of infection. Despite the severe symptoms and aggressive proliferation of BDV in the infected gerbils, no apparent neuroanatomical abnormalities or neuronal cell loss was observed in the infected gerbil brain. Furthermore, no or only minimal infiltration was observed in the infected gerbil brain. By in situ hybridization and real-time PCR analyses, we demonstrated that the predominant area of expression of BDV mRNA, as well as the protein, was shifted in the brain in association with progression of disease. In nondiseased gerbils, the virus replication was predominantly detected in the cerebral cortex and hippocampus of the CNS. On the other hand, diseased animals showed a high level of expression in the lower brain stem and cerebellum, especially in Purkinje cell neurons. These observations suggested that significant replication of the virus in specific areas of the CNS is critical for development of the neurological disorders in BDV-infected neonatal gerbils.
Asunto(s)
Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/patogenicidad , Encéfalo/virología , Enfermedades del Sistema Nervioso/virología , Animales , Animales Recién Nacidos , Western Blotting , Enfermedad de Borna/sangre , Gerbillinae , Inmunohistoquímica , Hibridación in Situ , Enfermedades del Sistema Nervioso/sangre , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Viral/análisis , Proteínas Virales/análisis , Proteínas Virales/genéticaRESUMEN
Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.
Asunto(s)
Virus de la Enfermedad de Borna/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Carioferinas , Proteínas de la Nucleocápside/metabolismo , Receptores Citoplasmáticos y Nucleares , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Células COS , Proteínas Portadoras/fisiología , Núcleo Celular/virología , Citoplasma/virología , Perros , Datos de Secuencia Molecular , Isoformas de Proteínas/fisiología , ARN Viral/metabolismo , Proteína Exportina 1RESUMEN
Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that belongs to the Mononegavirales. Unlike other animal viruses of this order, BDV replicates and transcribes in the nucleus of infected cells. Previous studies have shown that BDV uses RNA splicing machinery for its mRNA expression. In the present study, we identified spliced RNAs that use an alternative 3' splice site, SA3, in BDV-infected cell lines as well as infected animal brain cells. Transient transfection analysis of cDNA clones of BDV RNA revealed that although SA3 is a favorable splice site in mammalian cells, utilization of SA3 is negatively regulated in infected cells. This negative splicing activity of the SA3 site is regulated by a putative cis-acting region, the exon splicing suppressor (ESS), within the polymerase exon of BDV. The BDV ESS contains similar motifs to other known ESSs present in viral and cellular genes. Furthermore, our results indicated that a functional polyadenylation signal just upstream of the BDV ESS is also involved in the regulation of alternative splicing of BDV. These observations represent the first documentation of complex RNA splicing in animal RNA viruses and also provide new insight into the mechanism of regulation of alternative splicing in animal viruses.
Asunto(s)
Empalme Alternativo , Virus de la Enfermedad de Borna/genética , Regulación Viral de la Expresión Génica , ARN Viral , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Línea Celular , Perros , Genoma Viral , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Poli A , Ratas , Células Tumorales CultivadasRESUMEN
We developed the antigen capture enzyme-linked immunosorbent assay (ELISA) systems for quantification of Borna disease virus (BDV) major antigens, p40 and p24. Using these ELISAs, we quantified the two proteins in various BDV-infected materials, including the cell lysates and culture supernatants as well as the homogenates of experimental animal brains. The ELISAs were also applied to measure the infectious titer of BDV in persistently infected cell lines. Quantitative analysis with these ELISAs allowed us to measure the molecular ratio between the p40 and p24 in infected samples. Interestingly, the ratio of p24 to p40 in persistently infected cells was much higher than that observed in acutely infected cells although the ratios in the supernatants from both cell lines were quite similar. BDV-inoculated gerbil brain cells showed a relatively high ratio of p24 to p40 as compared with acutely infected cells. These observations suggested that the molecular ratio between the proteins strongly depended on the infectious status of BDV in the host cells. The ELISA system developed here could be a convenient method for the quantification of BDV infection and may also be beneficial for understanding viral replication and infectious status in the host cells.
Asunto(s)
Antígenos Virales/análisis , Virus de la Enfermedad de Borna/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Encéfalo/virología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Gerbillinae , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
We examined translational initiation of a bicistronic 0.8-kb mRNA of Borna disease virus (BDV) using a cDNA clone of the mRNA. Upon transfection with the clone, COS-7 cells produced a 16-kDa protein (P'), in addition to the previously identified products of BDV, 24- (P) and 14.5-kDa proteins. The 16-kDa product was detected by anti-P monoclonal antibody and was shown to exist in BDV-infected cell lines as well as in infected animal brain cells. Transient expression analysis of mutated cDNA clones encoding the BDV 0.8-kb mRNA revealed that the 16-kDa protein was initiated at the second AUG codon on the same open reading frame of the P protein. The mutational analysis also demonstrated that the first AUG within the 0.8-kb mRNA is not optimal, although the signal contains a better Kozak's motif. These results demonstrated the presence of three functional AUG codons in the smallest mRNA of BDV and also suggested that a leaky scanning mechanism is involved in translational initiation at AUG codons downstream of the bicistronic mRNA of BDV. Furthermore, the 16-kDa protein was located in the BDV-specific nuclear foci and was found to associate with the other viral proteins in BDV-infected cells, demonstrating an important role of the novel identified BDV protein in viral replication.
Asunto(s)
Virus de la Enfermedad de Borna/genética , Fosfoproteínas/genética , Biosíntesis de Proteínas , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Encéfalo/virología , Células COS , Línea Celular , Codón Iniciador , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Gerbillinae , Datos de Secuencia Molecular , Peso Molecular , Mutación , Operón , Iniciación de la Cadena Peptídica Traduccional/genética , Fosfoproteínas/análisis , Fosfoproteínas/biosíntesis , ARN Mensajero/genética , Transfección , Proteínas Virales/análisis , Proteínas Virales/química , Replicación ViralRESUMEN
A pregnant mare showing pyrexia, reduced appetite, ataxia and paresis was euthanized and examined for the presence of Borna disease virus (BDV). Her brain, showing multiple neuronal degeneration and necrosis with hemorrhage, and the histologically normal brain of the fetus were both positive for BDV RNA. The BDV nucleotide sequences were identical in the mare and fetus in the second open reading frame (ORF). This is the first report of the possible vertical transmission of BDV in a horse.
Asunto(s)
Enfermedad de Borna/transmisión , Virus de la Enfermedad de Borna/aislamiento & purificación , Enfermedades Fetales/virología , Enfermedades de los Caballos/transmisión , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Complicaciones Infecciosas del Embarazo/virología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/líquido cefalorraquídeo , Secuencia de Bases , Encéfalo/patología , Encéfalo/virología , Cartilla de ADN/química , ADN Viral/química , Femenino , Enfermedades de los Caballos/virología , Caballos , Immunoblotting/veterinaria , Inmunohistoquímica , Hibridación in Situ/veterinaria , Datos de Secuencia Molecular , Embarazo , ARN Viral/química , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
A total of 15 (T-1-T-15) domestic cats with neurological disorders in Tokyo area were examined for association with Borna disease virus (BDV). None had detectable antibodies to feline immunodeficiency virus (FIV), feline leukemia virus, feline infectious peritonitis virus and Toxoplasma gondii, and only cat T-8 had detectable antibody to FIV. Serological and molecular epidemiological studies revealed a significantly high prevalence of BDV infection in these cats: antibodies against BDV p24 and/or p40 proteins in 10/15 (66.7%) and p24 and/or p40 RNA in peripheral blood mononuclear cells in 8/15 (53.3%). Further, in situ hybridization and immunohistochemistry analyses of the autopsied brain samples derived from one of the cats (T-15) revealed BDV RNA predominantly in neuronal cells in restricted regions, such as olfactory bulb and medulla of cerebrum. Thus, BDV is present in Japanese domestic cats with neurological disorders at a high prevalence.