(1/250) Reverse transcription-nested polymerase chain reaction for detecting p40 RNA of Borna disease virus, without risk of plasmid contamination.
Several methods for the detection of Borna disease virus (BDV) RNA have been reported, one being the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method. However, due to the possibility of contamination of the cloned DNA in a reaction tube, false-positive results might be obtained by RT-nested PCR. To detect only BDV RNA without anxiety of contamination, we developed an RT-nested PCR system using "mRNA selective PCR kit". Using this system, cDNA of BDV p40 in the plasmid (up to 5 x 10(7) molecules) was not amplified. BDV specific sequence was amplified from total RNA (more than 50 pg) of MDCK/BDV cells, which were persistently infected with BDV. These results indicate that this mRNA selective RT-nested PCR system can specifically amplify target RNA as distinguished from plasmid contaminated. (+info)
(2/250) A serosurvey of Borna disease virus infection in wild rats by a capture ELISA.
For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV. (+info)
(3/250) Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells.
Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly up-regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors. (+info)
(4/250) Nuclear localization of the protein from the open reading frame x1 of the Borna disease virus was through interactions with the viral nucleoprotein.
Previous studies have predicted the presence of a small open reading frame (ORFx1) located between ORF-1 and ORF-2 of the Borna disease viral (BDV) genome. The ORFx1 is expressed as a p10 protein that is localized in the nucleus and cytoplasm of BDV-infected cells. In this study, we cloned the nucleotide sequence of ORFx1 into expression vectors and showed that it is expressed as p10. An anti-p10 serum gave nuclear and cytoplasmic staining of cells persistently infected with BDV. Immunoprecipitation of p10 from BDV-infected cells coprecipitated the p40 nucleoprotein N and the 24-kDa viral phosphoprotein P. Transient transfection of noninfected cells showed that p10 and p40 can be coprecipitated and revealed that p10 localized in the cytoplasm was imported into the nucleus in the presence of the BDV p40 N. In vitro protein-protein interaction studies on solid phase showed the direct interaction of the p10 with the BDV N protein. The subcellular distribution of p10 and its interaction with p40 suggest that this protein may play a role in the nuclear replication and/or transcription of BDV. (+info)
(5/250) Pathogenesis of borna disease virus: granulocyte fractions of psychiatric patients harbor infectious virus in the absence of antiviral antibodies.
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. (+info)
(6/250) Inhibition of Borna disease virus replication by ribavirin.
The guanosine analogue ribavirin was tested for antiviral activity in two neural cell lines, human oligodendrocytes and rat glia, against Borna disease virus (BDV) strains V and He/80. Ribavirin treatment resulted in lower levels of virus and viral transcripts within 12 h. Addition of guanosine but not adenosine resulted in a profound reduction of the ribavirin effect. Ribavirin appears to be an effective antiviral agent for treatment of BDV infection in vitro. A likely mechanism for its activity is reduction of the intracellular GTP pool, resulting in inhibition of transcription and capping of BDV mRNAs. (+info)
(7/250) T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein.
Infection of neonates with Borna disease virus (BDV) induces severe meningoencephalitis and neurological disorder in wild-type but not in beta(2)-microglobulin-deficient mice of strain MRL (H-2(k)). Temporary in vivo depletion of CD8(+) T cells delayed BDV-induced disease for several weeks. Depletion of CD4(+) T cells had a similar beneficial effect, indicating that the BDV-induced neurological disorder in mice is a CD4(+) T cell-dependent immunopathological process that is mediated by CD8(+) T cells. Lymphocytes prepared from brains of diseased mice were mainly from the CD8(+) T cell subset. They showed up-regulation of activation markers and exerted strong MHC I-restricted cytotoxic activity against target cells expressing the BDV nucleoprotein p40. Infection of B10.BR (H-2(k)) or congenic C57BL/10 (H-2(b)) mice resulted in symptomless, lifelong persistence of BDV in the brain. Superinfection with a recombinant vaccinia virus expressing BDV p40 but not with other vaccinia viruses induced severe neurological disease and encephalitis in persistently infected B10.BR mice but not in persistently infected C57BL/10 mice, indicating that the disease-inducing T cell response is restricted to the nucleoprotein of BDV in H-2(k) mice. Our results demonstrate that the cellular arm of the immune system may ignore the presence of a replicating virus in the central nervous system until proper antigenic stimulation at a peripheral site triggers the antiviral response. (+info)
(8/250) Enhancement of Borna disease virus transcription in persistently infected cells by serum starvation.
Transcription of Borna disease virus (BDV) in persistently infected MDCK (MDCK/BDV) cells increased in the fetal bovine serum free media as detected by Northern blot analysis. Especially, the amount of 1.9-kb RNA without cap formation at the 5' end and polyadenylation at the 3' end, increased as compared to other mRNA molecules of BDV. Growth arrest of MDCK/BDV cells observed in the condition of serum starvation might be important for increasing viral transcription. Since N-cadherin is the responsible factor for cell-to-cell contact, MDCK/BDV cells were cultured in calcium free medium which inhibits the interaction of N-cadherin. However, inhibition of cell-to-cell contact by N-cadherin is not effective on up regulation of viral transcription. Our finding in this study indicates that enhancement of BDV transcription by serum starvation is a useful technique for further investigation in understanding of mechanisms of BDV transcription. (+info)