Effect of Immune Priming on Borna Disease

Borna disease virus (BDV) is a neurotropic virus with a broad host and geographic range. Lewis rats were immunized against BDV with a recombinant vaccinia virus expressing the BDV nucleoprotein and were later infected with BDV to evaluate protection against Borna disease (BD). Relative to animals that were not immunized, immunized animals had a decreased viral burden after challenge with infectious virus, more marked inflammation, and aggravated clinical disease. These data suggest that a more robust immune response in Borna disease can reduce viral load at the expense of increased morbidity.     

A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-alpha is neither necessary nor sufficient

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-alpha has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO(-))-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-alpha. Moreover, the induction of TNF-alpha expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO(-). However, as opposed to the invading cells producing ONOO(-) that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO(-) is produced without pathological sequelae by IFN-gamma-stimulated neurovascular endothelial cells.     

Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.     

A Functional Role for Neutralizing Antibodies in Borna Disease: Influence on Virus Tropism outside the Central Nervous System

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.     

The peroxynitrite scavenger uric acid prevents inflammatory cell invasion into the central nervous system in experimental allergic encephalomyelitis through maintenance of blood-central nervous system barrier integrity

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO(-)), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO(-) or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO(-)-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO(-), or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.     

Neutralizing antibodies in persistent borna disease virus infection: prophylactic effect of gp94-specific monoclonal antibodies in preventing encephalitis

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.     

Downregulation of an astrocyte-derived inflammatory protein, S100B, reduces vascular inflammatory responses in brains persistently infected with Borna disease virus

Borna disease virus (BDV) is a neurotropic virus that causes a persistent infection in the central nervous system (CNS) of many vertebrate species. Although a severe reactive gliosis is observed in experimentally BDV-infected rat brains, little is known about the glial reactions contributing to the viral persistence and immune modulation in the CNS. In this regard, we examined the expression of an astrocyte-derived factor, S100B, in the brains of Lewis rats persistently infected with BDV. S100B is a Ca(2+)-binding protein produced mainly by astrocytes. A prominent role of this protein appears to be the promotion of vascular inflammatory responses through interaction with the receptor for advanced glycation end products (RAGE). Here we show that the expression of S100B is significantly reduced in BDV-infected brains despite severe astrocytosis with increased glial fibrillary acidic protein immunoreactivity. Interestingly, no upregulation of the expression of S100B, or RAGE, was observed in the persistently infected brains even when incited with several inflammatory stimuli, including lipopolysaccharide. In addition, expression of the vascular cell adhesion molecule 1 (VCAM-1), as well as the infiltration of encephalitogenic T cells, was significantly reduced in persistently infected brains in which an experimental autoimmune encephalomyelitis was induced by immunization with myelin-basic protein. Furthermore, we demonstrated that the continuous activation of S100B in the brain may be necessary for the progression of vascular immune responses in neonatally infected rat brains. Our results suggested that BDV infection may impair astrocyte functions via a downregulation of S100B expression, leading to the maintenance of a persistent infection.     

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6?weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.     

High-Dose Borna Disease Virus Infection Induces a Nucleoprotein-Specific Cytotoxic T-Lymphocyte Response and Prevention of Immunopathology

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.     

Pathogenic potential of borna disease virus lacking the immunodominant CD8 T-cell epitope

Borna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2(k) mice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2(k) mice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.     

Borna disease: association with a maturation defect in the cellular immune response.

Borna disease virus (BDV) is a negative-strand RNA virus which produces persistent infection in a variety of experimental animals. In the rat, the presence or absence of clinical signs of Borna disease, a characteristic, biphasic neurobehavioral illness, depends on host-related factors. A window of opportunity exists after birth wherein inoculation with BDV produces a persistently infected rat without signs of Borna disease or encephalitis (persistent, tolerant infection-newborn [PTI-NB] rat). Although immunopathological destruction of the nervous system does not occur in the PTI-NB rat, significant alterations in the development of the nervous system were noted, including site-specific lysis of neurons. Unlike the case with other pharmacologically produced, persistent, tolerant BDV infections, adoptive transfer of spleen cells from BDV-infected rats did not produce disease in the PTI-NB rats. PTI-NB rats developed Borna disease after being connected by parabiosis to rats with Borna disease. Bone marrow transplantation experiments revealed that bone marrow cells from PTI-NB rats produced Borna disease in lethally irradiated, BDV-infected recipient rats. Bone marrow from PTI-NB rats contained a complement of inflammatory cells capable of inducing Borna disease. Thus, the loss of BDV-specific cellular immunity appeared to occur after the release of cells from the bone marrow.     

Mutation of the Protein Kinase C Site in Borna Disease Virus Phosphoprotein Abrogates Viral Interference with Neuronal Signaling and Restores Normal Synaptic Activity

Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)–dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.     

MEK-specific inhibitor U0126 blocks spread of Borna disease virus in cultured cells

Borna disease virus (BDV) is a highly neurotropic virus that causes Borna disease, a virus-induced immune-mediated encephalomyelitis, in a variety of warm-blooded animals. Recent studies reported that BDV can be detected in patients with psychiatric disorders. BDV is noncytopathic, replicates in the nucleus of infected cells, and spreads intraaxonally in vivo. Upon infection of susceptible cultured cells, virus can be detected in foci. Little is known about the cellular components required for BDV replication. Here, we show that the cellular Raf/MEK/ERK signaling cascade is activated upon infection with BDV. In the presence of the MEK-specific inhibitor U0126, cells get infected with BDV; however, there is a block in virus spread to neighboring cells. The effect of the inhibitor on virus spread was still observed when the compound was added 2 h postinfection but not if treatment was initiated as late as 4 h after infection. Our results provide new insights into the BDV-host cell interaction and show that virus infection can be controlled with drugs interfering with a cellular signaling pathway. Since concentrations of the MEK inhibitor required to block BDV focus formation are not toxic for the host cells, our finding may be important with respect to antiviral drug development.     

Possible Correlation between Borna Disease Virus Infection and Japanese Patients with Chronic Fatigue Syndrome

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.     

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.     

Failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection

Rabies is a lethal disease caused by neurotropic viruses that are endemic in nature. When exposure to a potentially rabid animal is recognized, prompt administration of virus-neutralizing antibodies, together with active immunization, can prevent development of the disease. However, once the nonspecific clinical symptoms of rabies appear conventional postexposure treatment is unsuccessful. Over the last decade, rabies viruses associated with the silver-haired bat (SHBRV) have emerged as the leading cause of human deaths from rabies in the United States and Canada as a consequence of the fact that exposure to these viruses is often unnoticed. The need to treat SHBRV infection following the development of clinical rabies has lead us to investigate why the immune response to SHBRV fails to protect at a certain stage of infection. We have established that measurements of innate and adaptive immunity are indistinguishable between mice infected with the highly lethal SHBRV and mice infected with an attenuated laboratory rabies virus strain. While a fully functional immune response to SHBRV develops in the periphery of infected animals, the invasion of central nervous system (CNS) tissues by immune cells is reduced and, consequently, the virus is not cleared. Our data indicate that the specific deficit in the SHBRV-infected animal is an inability to enhance blood-brain barrier permeability in the cerebellum and deliver immune effectors to the CNS tissues. Conceivably, at the stage of infection where immune access to the infected CNS tissues is limited, either the provision or the development of antiviral immunity will be ineffective.