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In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
ABSTRACT
In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
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OBJECTIVE: During the last two decades, Borna disease virus (BDV) has received much attention as a possible zoonotic agent, particularly as a cause of psychiatric disease. Although several studies have shown that BDV is present in Asia, BDV has not been detected in Korea. This study was designed to further investigate the presence of BDV infection in Korea. METHODS: Blood samples were taken from 39 race horses and 48 jockeys. Antibody to BDV was detected by indirect immunofluorescence antibody test and RNA of BDV by real time reverse transcriptase PCR (rRT-PCR). RESULTS: No evidence of BDV was detected in either the horses or the jockeys group. CONCLUSION: Our results suggest that BDV infection may not be endemic in Korea. Further studies with novel diagnostic tools are required to clarify the prevalence of BDV infection in Korea.
Subject(s)
Animals , Humans , Asia , Borna Disease , Borna disease virus , Racial Groups , Fluorescent Antibody Technique, Indirect , Horses , Korea , Prevalence , Reverse Transcriptase Polymerase Chain Reaction , RNAABSTRACT
Objective To observe the epidemiology characterization of Borna disease virus (BDV) in animal brain in Ili, Xinjiang, and to find out the potential infection of the Borna disease virus to prevent its outbreak. Methods The BDV p24 gene of animal brain tissues in Ili including 200 horses, 75 donkeys and 100 shepherd dogs was detected by fluorescence quantitative nest reverse transcriptase polymer-ase chain reaction(FQ-nRT-PCR). GFP-p24,pMD-19 plasmid contamination was excluded from positive products. Clone sequencing was used to analyze the homology of gene and amino acid sequence. Results BDV p24 gent was found in 3 Ili horses, 4 Ili donkeys and 9 shepherd dogs, and the positive ratio is 1.5%, 5.3% and 9.0%, respectively. The GFP-p24,pMD-19 were not found in BDV p40 gene and plasmid stand-ard. The sequence of BDV p24 amplification production was totally the same as He/80 virus strain. Conclu-sion Natural infection of BDV may exist in the animals(horses, donkeys and dogs)in Ili, and the epidem-ic strain of BDV in this area was homological as He/80 virus strain.
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Objective To investigate the prevalence of Borna disease virus (BDV) in viral encephalitis patients in Ningxia Hui Autonomous Region and to analyze phylogenetic development of BDV in this area. Methods The gene fragments of BDV were amplified by real time nested reverse transcriptase polymerase chain reaction (PCR) from peripheral blood mononuclear cells (PBMC) of 60viral encephalitis (VE) patients. Enzyme linked immunosorbent assay (ELISA) was applied to detect p40 antibody in the cerebrospinal fluid from those patients who were confirmed as BDV p24 positive in previous study and those patients who were detected as BDV positive by PCR in the present study.The confirmed p40 positive samples were used for BDV gene cloning and sequencing, which were subsequently compared with the sequences of overseas standard strains, including He/80, H1766 and strain V by software MEGA and DnaSP4.0. The virus phylogenetic tree was constructed based on the above data. Results The positive rate of BDV p24 by PCR was 10. 08% (12/119) and the positive rate of BDV p40 by PCR was 5.88% (7/119). The cerebrospinal samples from two patients were detected as nucleoprotein antibody positive by ELISA, which resulted in a positive rate of 1.68%. The results of gene sequence analysis showed that the sequences of 6 cases among the 7 BDV positive patients were exactly same as the sequence of German horse-origin He/80 strain. Only one synonymous mutation was detected in the remaining one case. Reconstructed gene phylogenetic tree showed a hybrid China (Ningxia)- German and Japan branch was formed by the BDV stain isolated from VE patients in Ningxia and the overseas BDV standard stains. Conclusions BDV infection may exist in VE patients in Ningxia Hui Autonomous Region, which may be associated with close contact between human and animals. The gene sequence of stains isolated from patients in Ningxia is highly homological with the overseas standard strain, which suggests that the domestic BDV may come from overseas, while domestic strains with mutations couldn't be excluded.
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Objective To investigate the expression and location of Borna disease virus(BDV)nucleoprotein in transfected oligodendrocytes,and then to explore its expression mechanisms in oligodendroglial cell(OL)and their effects on cell proliferation.Methods We used PCR to detect BDVp40 gene fragments in transfected OL,laser confocal microscopy and Western blot method to detect the expression of nuclear protein and its intracellular location in the cell,MTT to detect the influence of cell proliferation by nuclear protein on the OL.Results We had detected the BDVp40 gene fragments in transfected oligodendrocytes.The nucleoprotein can be positioned in the cytoplasm and cell membrane by laser scanning confocal microscope;Western blot results showed that there is nucleoprotein in the constitutive protein,but was not detected in the cytoplasm.MTT tests showed that nucleoprotein expressed in OL can inhibit cell proliferation.Conclusion It is indicated that the transfected OL stablely express of BDV nucleoprotein,and itslocation is in the cell structure of proteins,particularly in the cytoplasm and more richer in cell membrane,that indicating they may play a key role in cell signal transduction or into the cell-mediated viral infection.BDV nucleoprotein inhibit proliferation of OL,which maybe an important mechanism of Borna disease virus persistant infection and produce symptoms.
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Objective To gain detailed insights into the Borna disease virus infection and its genetic characteristics and phylogeny in Ningxia,China.Methods BDV p24 segment were detected by fluorescence quantitative nested RT-PCR from peripheral blood mononuclear cells of 119 patients with viral encephalitis,205 cattle,978 sheep,46 patients with cerebrovascular diseases and 13 patients with multiple sclerosis.Data from phylogenetic analysis on BDV p24 positive samples together with those from the positive gene sequences of animals and patients in the previous studies in Ningxia,were compared to the 29 sequences provided by the GenBank from 7 animals in 5 countries.Both the sequence homologous similarity of the nucleotide and amino acid sequences were analyzed and gene phylogenetic tree was reconstructed.Results Data from 23 collections of positive test samples,together with the ones from early detection of gene sequence analysis,showed that the homologous similarity sequences of both nucleotide and amino acid was between 95.3% and 100.0% and highly homophylic with HE80 that detected from ill horses in Germany.One part of nucleotide sequences formed the 'Ningxia independent branch'while the other one belonged to the 'Germany-Ningxia-Japan mixed branch'.There was a high identity within the branch.Conclusion A Ningxia independent BDV strain from geographical origin might exist while the epidemic strains were imported with multiple sources.
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The biological cause of psychiatric illnesses continues to be under intense scrutiny. Among the various neurotropic viruses, Borna disease virus (BDV) is another virus that preferentially targets the neurons of the limbic system and has been shown to be associated with behavioural abnormalities. Presence of various BDV markers, including viral RNA, in patients with affective and mood disorders have triggered ongoing debate worldwide regarding its aetiopathogenic relationship. This article analyses its current state of knowledge and recent advances in diagnosis in order to prove or refute the association of BDV in causation of human neuropsychiatric disorders. This emerging viral causative association of behavioural disorders, which seems to be inching closer, has implication not only for a paradigm shift in the treatment and management of neuropsychiatric illnesses but also has an important impact on the public health systems.
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Objective To investigate the epidemioiogical pattern of Borna disease virus (BDV) among different canine breeds in Ili, China, and to analyze its potential phylogeny. Methods BDV p24 RNA fragments were detected from peripheral blood mononuclear cells (PBMCs) of canine by modified nested RT-PCR (nRT-PCR). Possible false positives were excluded by determination of both BDV p40 RNA fragments and PMD19 plasmid standards. Analysis were performed on genetic sequence, homologous comparison, amino acid sequence and phylogeny after p24 positive products were validated. Results BDV p24 RNA fragments were found only in Kazakh Tobet (a shepherd dog) in 8 breeds of 150 cases and their overall positive rate was 11.0% (10/91). Compared with the strain of He/80 from horse and that of S6 from sheep in Germany, the homologous similarities of Kazakh Tobet was 99.2% and 95.7%, and that of amino acid as 100% and 89.3%, respectively. The kinship of Kazakh Tobet was close to He/80 and next to S6. Conclusion There was potential natural BDV infection in Kazakh Tobet in Ili, and its endemic strain was concerned with He/ 80 infecting Ili horse and S6 of German Merino sheep introduced into the region from Germany.
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OBJECTIVE: Borna disease virus (BDV) is a highly neurotropic agent causing various neuropsychiatric symptoms in animals. Over the past two decades, it has been suggested that BDV might be associated with human psychiatric diseases. We aimed to investigate whether BDV is associated with psychiatric patients in Korea. METHODS: We recruited 60 normal controls and 198 psychiatric patients (98 patients with depressive disorder, 60 with schizophrenia, and 40 with bipolar disorder). We used an indirect immunofluorescence antibody (IFA) test for the BDV antibody and a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay for p24 and p40 RNA from peripheral blood mononuclear cells (PBMCs). RESULTS: Neither the BDV antibody nor p24, p40 RNA was detected in controls and patients groups. CONCLUSION: Our results suggest that BDV might not be associated with psychiatric patients in Korea.
Subject(s)
Animals , Humans , Borna Disease , Borna disease virus , Corynebacterium , Depressive Disorder , Fluorescent Antibody Technique, Indirect , Korea , Reverse Transcriptase Polymerase Chain Reaction , RNA , SchizophreniaABSTRACT
OBJECTIVE: Borna disease virus (BDV) is a highly neurotropic agent causing various neuropsychiatric symptoms in animals. Over the past two decades, it has been suggested that BDV might be associated with human psychiatric diseases. We aimed to investigate whether BDV is associated with psychiatric patients in Korea. METHODS: We recruited 60 normal controls and 198 psychiatric patients (98 patients with depressive disorder, 60 with schizophrenia, and 40 with bipolar disorder). We used an indirect immunofluorescence antibody (IFA) test for the BDV antibody and a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay for p24 and p40 RNA from peripheral blood mononuclear cells (PBMCs). RESULTS: Neither the BDV antibody nor p24, p40 RNA was detected in controls and patients groups. CONCLUSION: Our results suggest that BDV might not be associated with psychiatric patients in Korea.
Subject(s)
Animals , Humans , Borna Disease , Borna disease virus , Corynebacterium , Depressive Disorder , Fluorescent Antibody Technique, Indirect , Korea , Reverse Transcriptase Polymerase Chain Reaction , RNA , SchizophreniaABSTRACT
Objective:It intended to examine whether there is BDV infection in the human tumor tissues of central nervous system in China and investigate the correlation between BDV infection and tumom of central nervous system.Methods:Nested reverse transcriptase polymerase chain reaction(nRT-PCR)and fluorescence quantitative polymerase chain reaction(FQ-PCR)was used to detect the BDV p24 fragments in 60 samples of human tumor tissues of central nervous system and 14 normal brain tissues.Results:The study indicated the positive rate of the BDV p24 fragment in human tumor tissues of the central nervous system (6.67%)was higher than that in normal brain tissues(0),but no statistical significance(P>0.05).Concluswn:It suggests that the BDV infection is present in the human tumor tissues of central nervous system in China.while the sample size wa.sn't large enough and we could not certify the possible correlation between BDV infection and cenfral nervous system tumors.
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Objective To explore the relationship between viral encephalitis(VE)and Borna disease virus(BDV)infection,and discuss the clinical features of viral encephalitis patients infected by BDV.Methods The p24 gene fragment of BDV in cerebrospinal fluid mononuclear cells(CSFMC)from 32 patients with viral encephaliris and 34 healthy individuals were examined by fluorescence quantitative nested reverse transcriptase polymerase chain reaction(FQ-nRT-PCR),in which, β-actin was used as internal reference. The clinical manifestations of patients with positive BDV p24 were watched. And the gene sequence,homology and amino acid sequence of positive products were analyzed. Results The positive rate(4/32,12.5%)of BDV p24 in CSFMC of patients with viral encephalitis was significantly higher than that of healthy individuals(0/34,0%),P＜0.05,and the numbers of copies of BDV p24 in CSFMC of VE patients were more than 102/μl. Compared with the sequences of the standard strain Ⅴ and the virus strain H1766 of BDV from horse supplied by GenBank,the homology of the gene sequence of positive product of CSFMC was 95.35% and 98.84% with gene mutation in 4 sites(nt1649 T→C, nt1656 G→A,nt1670 C→T, nt1676 C→T). The genetic relationship between the positive product and the virus strain BDV from horse was the nearest. The clinical features of VE patients with positive BDV p24 were mainly by mental and behavior disorders. Conclusion The occurrence of VE patients in the city of Zunyi in Guizhou province may be associated with BDV infection. The clinical features of VE patients with positive BDV p24 were mainly mental and behavior disorders.
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Objective In order to study the epidemiology of Borna disease virus (BDV) in Zunyi region and its surrounding regions in Guizhou province. Methods p24 fragment of BDV fragments in peripheral blood mononuclear cells (PBMC) from 43 patients with viral encephalitis (VE), 9 cases with multiple sclerosis (MS), 7 cases with Guillain-Barre syndrome (GBS), 5 cases with Parkinson disease (PD), 98 healthy donors and 300 goats were examined by quantitative fluorescence nested reverse transcriptase polymerase chain reaction (PCR). Gene sequence and amino acid sequence were analyzed for positive products. Results The positive rate of BDV p24 fragment in PBMC from VE (13.95 %) and MS (22.22 %) were significantly higher than in healthy donors (0 %, P<0.05). The positive rate of BDV p24 fragment in PBMC from goats was 0.67 %, without statistical difference when compared with healthy donors (P>0.05). Guillain-Barre syndrome and Parkinson disease( PD)were tested negative. The sequence of the BDV p24 fragment from the patients with VE was in conformity with that of the MS. Results presented that 3 situs consistency silent mutation when compared with strain V and its homogeneity was 96.51%. 2 situs consistency silent mutation compared with BDV/MDCK and its homogeneity was 97.67%. 2 situs consistency silent mutation when compared with C6BV and its homogeneity was 97.67 %. Sequences of the BDV p24 fragment from the goats presented 3 situs consistency silent mutation when compared with strain V and its homogeneity was 96.51%. 3 situs consistency silent mutation when compared with BDV/MDCK and its homogeneity was 96.51%. 3 situs consistency silent mutation when compared with C6BV and its homogeneity was 96.51%. However, there were no changes of encoding amino acids in all BDV p24 fragments from neuropsychiatric disorders. Conclusion Our data indicated that the BDV infection was presented in patients with VE, MS and goats from Zunyi region and its surrounding regions of Guizhou province. BDV might play a potential role in the development of VE, MS as well as having correlations with animals.
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Objective To investigate the epidemiological pattern of Borna disease virus (BDV)infection in horses and to analyze the phylogenetic tree of derived BDV in Yili, Xinjiang. Methods We established a modified nested RT-PCR (nRT-PCR) to detect BDV p24 segment in peripheral blood mononuclear cells (PBMCs) and brain tissues of 120 horses in Yili, Xinjiang. Positive products were analyzed by sequencing and homology analysis. Results The positive rate of BDV infection was 2.5% in both PMBCs and brain tissues at the same time. The gene sequence revealed in positive PCR samples was more than 93 % ,identical to that of BDV derived from horses in other countries. We also noticed a high degree of identity ( >98 % ) to standard strain He/80 in gene sequence of positive PCR samples. Conclusion Our study found the presence of BDV natural infection in horses in Yili. The endemic BDV had a high degree of identity to standard strain He/80.
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Objective:To establish fluorescence quantitative nested RT-PCR method for detecting Borna disease virus (BDV).Methods:According to the specific sequence of BDV P24 genes,the primers and the fluorescence probe were designed and synthesized.The fragment generated by PCR was cloned into the pMD18-T vector.The positive recombinant plasmid would be used as standard quantitative template to make the standard curve for sample detection.Results:The standard curve indicated the linear relationship between Ct(cycle threshold) and template concentration (r=0.998).The fluorescence quantitative nested RT-PCR method for detection of BDV p24 fragment was established.And it was used to detect BDV p24 fragment in peripheral blood mononuclear cells(PBMC)from 58 patients with neuropsychiatric disorders and 50 healthy blood donors.4 patients with neuropsychiatric disorders were positive,and normal control negative.Conclusion:The fluorescence quantitative nested RT-PCR method for detection of Borna disease virus can eliminate PCR cross-contamination which causes false positive,and real-time detection ensures accurate quantity.It can be used to study the association between BDV infection and human neuropsychiatric disorders.
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Objective In order to investigate the relationship between Borna disease virus(BDV) and the viral encephalitis,and whether BDV infection exists in Ningxia or not.Methods The p24 fragment of BDV in peripheral blood mononuclear cells(PBMC) and cerebrospinal fluid mononuclear cells(CSFMC) in 52 patients with viral encephalitis and 32 healthy donors were examined by nested reverse transcriptase polymerase chain reaction(RT-PCR) and fluorescence quantitative(FQ) PCR to analysis gene sequence,homogenicity and amino acid sequence of positive products.Results The positive rate of BDV p24 in PBMC and CSFMC in viral encephalitis(9.6%,11.5%) was significantly higher than that in healthy donors(0,P
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ObjectiveTo study the relationship between Borna disease virus (BDV) and the human viral encephalitis.MethodsThe P24 fragment of BDV RNA in peripheral blood mononuclear cells (PBMC) from 59 patients with viral encephalitis diagnosed clinically, and 112 healthy donors were examined by fluorescence quantitative nested reverse transcriptase polymerase chain reaction (FQ-nRT-PCR).ResultsThere were 3 positive of BDV P24 fragment in 59 patients with viral encephalitis, and no positive in blood donors. The positive rate of BDV p24 in PBMC in viral encephalitis (5.08%) was higher significantly than that in blood donors (P