Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections.

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.

Application of antibodies against Borna disease virus phosphoprotein and nucleoprotein on paraffin sections.

In order to study the application of antibodies against recombinant proteins for detecting Borna disease virus (BDV) phosphoprotein (p24) and nucleoprotein (p40) (BDV­p24/p40) on paraffin sections by immunohistochemistry. The purified fusion p24 and p40 proteins were used for the preparation of polyclonal and monoclonal anti­p24 and anti­40 antibodies, which were confirmed by ELISA and western blotting. Paraffin sections were made from BDV­infected Sprague­Dawley (SD) rats (n=20), PBS­injected SD rats (n=20), normal SD rats (n=20) and normal C57 mice (n=20). Immunohistochemical staining was performed according to the EnVision™ two­step protocol. Heat­mediated antigen retrieval was performed using the retrieval buffer sodium citrate (1 mM; pH 6.0). All the antibodies against recombinant proteins exhibited good sensitivity and specificity. There were significant differences between the BDV­infected group and the BDV­uninfected group for poly­ and monoclonal anti­p24 and ­p40 antibodies. These antibodies against recombinant proteins may be used effectively to detect BDV p24 and p40 in paraffin sections.

Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels.

We have demonstrated that vaccination of cockatiels (Nymphicus hollandicus) with killed parrot bornavirus (PaBV) plus recombinant PaBV-4 nucleoprotein (N) in alum was protective against disease in birds challenged with a virulent bornavirus isolate (PaBV-2). Unvaccinated birds, as well as birds vaccinated after challenge, developed gross and histologic lesions typical of proventricular dilatation disease (PDD). There was no evidence that vaccination either before or after challenge made the infection more severe. Birds vaccinated prior to challenge largely remained free of disease, despite the persistence of the virus in many organs. Similar results were obtained when recombinant N, in alum, was used for vaccination. In some rodent models, Borna disease is immune mediated thus we did an additional study whereby cyclosporine A was administered to unvaccinated birds starting 1day prior to challenge. This treatment also conferred complete protection from disease, but not infection.

Change in the responsiveness of interferon-stimulated genes during early pregnancy in cows with Borna virus-1 infection.

BACKGROUND: Borna disease virus is a neurotropic pathogen and infects the central nervous system. This virus infected a variety of animal species including cows. The most of cows infected with Borna disease virus 1 (BoDV-1) exhibit subclinical infection without any neurological symptoms throughout their lifetime. We previously reported on the low conception rates in-seropositive cows. Interferon-τ (IFN-τ) plays an important role in stable fertilization, and is produced from the fetal side following embryo growth at 15-40 days of pregnancy. IFN-τ induces the expression of interferon-stimulated gene (ISG) 15 and Mx2 in peripheral blood mononuclear cells (PBMCs). To understand the embryo growth and maternal reaction during early pregnancy in cows with BoDV-1 infection, we aimed to assess the gene expression of ISG15 and Mx2 from PBMCs in BoDV-1-seropositive cows. RESULTS: None of the cows showed any clinical and neurological symptoms. Among the cows that conceived, the expressions of the ISG15 and Mx2 genes were greater in the BoDV-1-seropositive cows than in the BoDV-1-seronegative cows; the difference was significant between the cows that conceived and those that did not (P < 0.05). CONCLUSIONS: The expression of ISG15 and Mx2 genes during early pregnancy significantly increased in the BoDV-1-seropositive cows and may be important for the maintenance of stable pregnancy in BoDV-1-infected cows. In contrast, the gene expression levels of ISG15 and Mx2 did not significantly increase during early pregnancy in BoDV-1-seronegative cows. Thus, BoDV-1 infection may lead to instability in the maintenance of early pregnancy by interfering with INF-τ production.

Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue.

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.

Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155.

It has been reported that the Borna disease virus (BDV) encoded phosphoprotein (P protein) can inhibit the activity of Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK-1), thus preventing the induction of type I interferon (IFN). However, the effects of microRNA on the regulation of BDV infection and the host's immune response have not been characterized. miR-155 was predicted to be complementary to the BDV P mRNA by RNAhybrid software. Here, we showed that miR-155 was down-regulated in BDV persistently infected human oligodendroglial (OL/BDV) cells and that the BDV P protein, but not the X protein, directly inhibited miR-155 expression in cells. When miR-155 was over-expressed, the inhibition of type I IFNs by BDV in cells was reversed, and the expression of type I IFNs was increased. When miR-155 expression was specifically blocked, cellular IFN expression and the induction of IFN by poly I:C treatment were suppressed. Furthermore, miR-155 promoted type I IFN production by targeting suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Mutations in the nt1138-nt1158 region of SOCS3 abandoned the impact of miR-155 on the expression of SOCS3-enhanced green fluorescent protein (EGFP). The levels of BDV P mRNA and protein were significantly decreased in OL/BDV cells when miR-155 was over-expressed; however, miR-155-mutation did not affect the expression of BDV P-EGFP. Thus, BDV persistent infection inhibited the expression of type I IFNs through the suppression of miR-155, and miR-155 played an important immune regulatory role in BDV persistent infection.

Serological evidence for infections with Borna disease virus in Turkey.

Distribution of Borna disease virus (BDV) infection outside endemic areas has been studied in several countries. We examined serum samples for anti-BDV antibodies in purebred racing horses and other domestic animals in Turkey. In total serum samples of 437 animals including 282 horses, 50 sheep, 25 goats, 50 cattle, and 30 cats were tested by indirect immunofluorescence assay (IFA). Anti-BDV antibodies were detected in 4.9% of horses, 12% of sheep, 4% of goats, 14% of cattle and 6.6% of cats. No statistical difference was observed between seroprevalence in Arabic and English purebred horses from four different racing centers (p > 0.05). Antibody titers ranged between 1:10 and 1:320. The highest antibody titers were found in sheep and horses and the lowest titer in cattle. Clinical symptoms of Borna disease were not observed in any animal of any species examined. This study confirms the presence of anti-BDV antibodies in racing horses as well as cat population in Turkey. Moreover anti-BDV antibodies are demonstrated for the first time in sheep, goats and cattle in Turkey.

Markers of Borna disease virus infection in cats with staggering disease.

Borna disease virus (BDV) is a RNA-virus causing neurological disorders in a wide range of mammals. In cats, BDV infection may cause staggering disease. Presently, staggering disease is a tentative clinical diagnosis, only confirmed at necropsy. In this study, cats with staggering disease were investigated to study markers of BDV infection aiming for improvement of current diagnostics. Nineteen cats fulfilled the inclusion criteria based on neurological signs and pathological findings. In 17/19 cats, BDV infection markers (BDV-specific antibodies and/or BDV-RNA) were found, and antibodies in serum (13/16, 81%) were the most common marker. BDV-RNA was found in 11/19 cats (58%). In a reference population without neurological signs, 4/25 cats were seropositive (16%). The clinical history and neurological signs in combination with presence of BDV infection markers, where serology and rRT-PCR on blood can be helpful tools, improve the diagnostic accuracy in the living cat.

The influence of Borna disease viral infection on dairy cow reproduction.

We investigated the influence of Borna disease virus (BDV) infection on the clinical state of dairy cows. Sera from 149 cows were examined using enzyme-linked immunosorbent assay and western blotting detect antibodies to the BDV-nucleoprotein antigen. Among 149 investigated cows, 25 (16.8%) showed a positive reaction to BDV antigen. No significant difference existed in milk production or medical history between seropositive and seronegative cows. Although the estrus cycle appeared normal even in the seropositive cows, the frequency of artificial insemination and calving-to-conception intervals significantly increased in seropositive cows. Therefore, fertilization failure was recognized in the BDV-antibody positive cows.

Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection.

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage.

Intracerebral Borna disease virus infection of bank voles leading to peripheral spread and reverse transcription of viral RNA.

Bornaviruses, which chronically infect many species, can cause severe neurological diseases in some animal species; their association with human neuropsychiatric disorders is, however, debatable. The epidemiology of Borna disease virus (BDV), as for other members of the family Bornaviridae, is largely unknown, although evidence exists for a reservoir in small mammals, for example bank voles (Myodes glareolus). In addition to the current exogenous infections and despite the fact that bornaviruses have an RNA genome, bornavirus sequences integrated into the genomes of several vertebrates millions of years ago. Our hypothesis is that the bank vole, a common wild rodent species in traditional BDV-endemic areas, can serve as a viral host; we therefore explored whether this species can be infected with BDV, and if so, how the virus spreads and whether viral RNA is transcribed into DNA in vivo.We infected neonate bank voles intracerebrally with BDV and euthanized them 2 to 8 weeks post-infection. Specific Ig antibodies were detectable in 41%. Histological evaluation revealed no significant pathological alterations, but BDV RNA and antigen were detectable in all infected brains. Immunohistology demonstrated centrifugal spread throughout the nervous tissue, because viral antigen was widespread in peripheral nerves and ganglia, including the mediastinum, esophagus, and urinary bladder. This was associated with viral shedding in feces, of which 54% were BDV RNA-positive, and urine at 17%. BDV nucleocapsid gene DNA occurred in 66% of the infected voles, and, surprisingly, occasionally also phosphoprotein DNA. Thus, intracerebral BDV infection of bank vole led to systemic infection of the nervous tissue and viral excretion, as well as frequent reverse transcription of the BDV genome, enabling genomic integration. This first experimental bornavirus infection in wild mammals confirms the recent findings regarding bornavirus DNA, and suggests that bank voles are capable of bornavirus transmission.

Expression of interferon gamma in the brain of cats with natural Borna disease virus infection.

Borna disease virus (BDV) is a neurotropic, negative-stranded RNA virus, which causes a non-suppurative meningoencephalomyelitis in a wide range of animals. In cats, BDV infection leads to staggering disease. In spite of a vigorous immune response the virus persists in the central nervous system (CNS) in both experimentally and naturally infected animals. Since the CNS is vulnerable to cytotoxic effects mediated via NK-cells and cytotoxic T-cells, other non-cytolytic mechanisms such as the interferon (IFN) system is favourable for viral clearance. In this study, IFN-γ expression in the brain of cats with clinical signs of staggering disease (N=12) was compared to the expression in cats with no signs of this disease (N=7) by quantitative RT-PCR. The IFN-γ expression was normalised against the expression of three reference genes (HPRT, RPS7, YWHAZ). Cats with staggering disease had significantly higher expression of IFN-γ compared to the control cats (p-value ≤ 0.001). There was no significant difference of the IFN-γ expression in BDV-positive (N=7) and -negative (N=5) cats having clinical signs of staggering disease. However, as BDV-RNA still could be detected, despite an intense IFN-γ expression, BDV needs to have mechanisms to evade this antiviral immune response of the host, to be able to persist.

Borna disease virus (BDV) circulating immunocomplex positivity in addicted patients in the Czech Republic: a prospective cohort analysis.

BACKGROUND: Borna disease virus (BDV) is an RNA virus belonging to the family Bornaviridae. Borna disease virus is a neurotropic virus that causes changes in mood, behaviour and cognition. BDV causes persistent infection of the central nervous system. Immune changes lead to activation of infection. Alcohol and drug dependence are associated with immune impairment. METHODS: We examined the seropositivity of BDV circulating immunocomplexes (CIC) in patients with alcohol and drug dependence and healthy individuals (blood donors). We examined 41 addicted patients for the presence of BDV CIC in the serum by ELISA at the beginning of detoxification, and after eight weeks of abstinence. This is the first such study performed in patients with alcohol and drug dependence. RESULTS: BDV CIC positivity was detected in 36.59% of addicted patients on day 0 and in 42.86% on day 56. The control group was 37.3% positive. However, we did not detect higher BDV CIC positivity in addicted patients in comparison with blood donors (p = 0.179). The significantly higher level of BDV CIC was associated with lower levels of GGT (gamma glutamyl transferase) (p = 0.027) and approached statistical significance with the lower age of addicted patients (p = 0.064). We did not find any association between BDV CIC positivity and other anamnestic and demographic characteristics. CONCLUSIONS: In our study addicted patients did not have significantly higher levels of BDV CIC than the control group. The highest levels of BDV CIC were detected in patients with lower levels of GGT and a lower age. TRIAL REGISTRATION: This study was approved by the ethical committee of the University Hospital Medical Faculty of Charles University in Pilsen, Czech Republic (registration number 303/2001).

Anti-Borna disease virus antibody responses in psychiatric patients: long-term follow up.

AIM: Data suggesting a pathogenetic role for Borna disease virus (BDV) in neuropsychiatric diseases are still inconclusive and it is unknown whether humans become persistently infected or clear the virus infection. The aim of the present study was therefore to investigate long-term BDV-specific antibody responses in psychiatric patients in order to gain new insights into human BDV infection and its pathogenicity. METHODS: BDV-specific antibody titers and associations with clinical conditions were studied retrospectively in 94 seropositive patients with schizophrenia (n = 46), affective disorders (n = 19) and other psychiatric disorders (n = 29) who had been repeatedly tested for the presence of BDV-specific antibodies on indirect immunofluorescence assay between 1985 and 2006. Long-term titer dynamics were studied in 46 patients followed up for a period of >36 months. RESULTS: A total of 25 of these 46 patients (54.3%) had persistent seropositivity, whereas seroreversion from positive to negative was observed in 21 (45.7%). Patients in the early course of schizophrenia had lower antibody titers compared to patients in the advanced course (P = 0.017), while a higher proportion of patients in the early course had titer increases (P < 0.05). There were no significant differences in antibody titers between patient subgroups with clinically stable and acute psychiatric disorders. CONCLUSION: Persistent seropositivity in a subgroup of psychiatric patients in the long-term analysis suggests chronic BDV infection in humans.

Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species.

Borna disease is a severe viral-induced disorder of the central nervous system of horses, sheep, and a few other animal species, occurring in certain areas of central Europe. Pathogenesis and epidemiology of natural Borna disease virus (BDV) infections are still not fully understood; several unique epidemiologic features, however, point toward the existence of BDV reservoir populations other than the final hosts. In this study, 69 mice and 12 shrews were trapped and examined. The virus distribution was investigated in detail in 2 BDV-positive bicolored white-toothed shrews, Crocidura leucodon, by immunohistochemistry and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). RT-PCR amplification products were sequenced, and the sequences were compared. These shrews had been collected in a BDV-endemic geographical region using live traps and did not show obvious clinical or pathological disease signs. BDV antigen and nucleic acid were identified in several organs, including the brain, mainly in nerve tissue and neurons, respectively, but also in parenchymal cells (eg, hepatocytes, Leydig cells) and epithelial cells, particularly of the respiratory and urogenital tract.

Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection.

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to certain neuronal populations. Since persistent BDV infection of neurons is nonlytic in vitro, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brains remain unclear. Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia. Here, we evaluated the mechanisms whereby astrocytes can contribute to activation of microglia in neuron-glia-microglia mixed cultures. We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES. Activation of astrocytes then produces activation of microglia as evidenced by increased formation of round-shaped, MHCI-, MHCII- and IL-6-positive microglia cells. Our analysis of possible molecular mechanisms of activation of astrocytes and/or microglia in culture indicates that the mediators of activation may be soluble heat-resistant, low molecular weight factors. The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons. The data are consistent with the hypothesis that microglia activation in the absence of neuronal damage may represent initial steps in the gradual neurodegeneration observed in brains of neonatally BDV-infected rats.

Isotype analysis of human anti-Borna disease virus antibodies in Japanese psychiatric and general population.

BACKGROUND: Borna disease virus (BDV) infection of humans has not been widely accepted due to the low titer of the antibody and lack of reliable diagnostic tools. OBJECTIVES: To examine exposure to BDV or a related virus in Japanese psychiatric and general population by detecting the specific IgG, IgM, and IgA. STUDY DESIGN: Sera from 304 psychiatric and 378 control subjects were examined for IgG, IgM, and IgA against BDV nucleoprotein (N) and phosphoprotein (P) using highly specific and sensitive radioligand assay. The avidity was also examined. RESULTS: The specific IgG, IgM, and IgA against both BDV-N and -P were detected and the seropositivity was not significantly different between patients and controls. The avidity of the specific IgG was low to moderate, and the specific IgM did not disappear for several years. CONCLUSIONS: Our results suggested common exposure to BDV or a related virus in the general Japanese population. Low avidity IgG and persistent IgM suggested delayed immune response against BDV or a related virus. The specific IgA indicated mucosal involvement.

Persistent Borna Disease Virus (BDV) infection activates microglia prior to a detectable loss of granule cells in the hippocampus.

Neonatal Borna Disease Virus (BDV) infection in rats leads to a neuronal loss in the cortex, hippocampus and cerebellum. Since BDV is a non-lytic infection in vitro, it has been suggested that activated microglia could contribute to neuronal damage. It is also conceivable that BDV-induced cell death triggers activation of microglia to remove cell debris. Although an overall temporal association between neuronal loss and microgliosis has been demonstrated in BDV-infected rats, it remains unclear if microgliosis precedes or results from neuronal damage. We investigated the timing of microglia activation and neuronal elimination in the dentate gyrus (DG) of the hippocampus. We found a significant increase in the number of ED1+ microglia cells as early as 10 days post infection (dpi) while a detectable loss of granule cells of the DG was not seen until 30 dpi. The data demonstrate for the first time that a non-lytic persistent virus infection of neurons activates microglia long before any measurable neuronal loss.

CD8 T cells require gamma interferon to clear borna disease virus from the brain and prevent immune system-mediated neuronal damage.

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.