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.     

Borna disease virus: a unique pathogen and its interaction with intracellular signalling pathways

Borna disease virus (BDV) is a neurotropic RNA virus that establishes non-cytolytic persistent infection in the central nervous system of warm-blooded animals. Depending on the host species and the route of infection, BDV persistence can modulate neuronal plasticity and animal behaviour and/or may provoke a T cell-mediated immunopathological reaction with high mortality. Therefore, BDV functions as a model pathogen to study persistent virus infection in the central nervous system. Here, we review recent evidence showing that BDV interferes with a spectrum of intracellular signalling pathways, which may be involved in viral spread, maintenance of persistence and modulation of neurotransmitter pathways.     

Two major histocompatibility complex class I-restricted epitopes of the Borna disease virus p10 protein identified by cytotoxic T lymphocytes induced by DNA-based immunization

Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the L-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M(1)SSDLRLTLL(10) and T(8)LLELVRRL(16), associated with the RT1.A(l) major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.A(l)-restricted A(230)SYAQMTTY(238) peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.     

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.     

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.     

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.     

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.     

Aggravation of viral hepatitis by platelet-derived serotonin

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8(+) T cell-dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8(+) T cell-dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8(+) T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.     

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.     

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.     

CD8(+) T lymphocytes mediate Borna disease virus-induced immunopathology independently of perforin

Perforin-mediated lysis of target cells is the major antiviral effector mechanism of CD8(+) T lymphocytes. We have analyzed the role of perforin in a mouse model for CD8(+) T-cell-mediated central nervous system (CNS) immunopathology induced by Borna disease virus. When a defective perforin gene was introduced into the genetic background of the Borna disease-susceptible mouse strain MRL, the resulting perforin-deficient mice developed strong neurological disease in response to infection indistinguishable from that of their perforin-expressing littermates. The onset of disease was slightly delayed. Brains of diseased perforin-deficient mice showed similar amounts and a similar distribution of CD8(+) T cells as wild-type animals. Perforin deficiency had no impact on the kinetics of viral spread through the CNS. Unlike brain lymphocytes from diseased wild-type mice, lymphocytes from perforin-deficient MRL mice showed no in vitro cytolytic activity towards target cells expressing the nucleoprotein of Borna disease virus. Taken together, these results demonstrate that CD8(+) T cells mediate Borna disease independent of perforin. They further suggest that the pathogenic potential of CNS-infiltrating CD8(+) T cells does not primarily reside in their lytic activity but rather in other functions.     

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.     

Borna disease virus nucleoprotein interacts with the CDC2-cyclin B1 complex

Transition from G(2) to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G(2)-to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.     

Borna Disease Virus-Induced Neurological Disorder in Mice: Infection of Neonates Results in Immunopathology

Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that persistently infects warm-blooded animals. In horses and other natural animal hosts, infections with BDV cause meningoencephalitis and behavioral disturbances. Experimental infection of adult mice takes a nonsymptomatic course, an observation previously believed to indicate that this animal species is not suitable for pathogenesis studies. We now demonstrate that BDV frequently induces severe neurological disease in infected newborn mice. Signs of neurological disease were first observed 4 to 6 weeks after intracerebral infection. They included a characteristic nonphysiological position of the hind limbs at an early stage of the disease and paraparesis at a later stage. Histological examination revealed large numbers of perivascular and meningeal inflammatory cells in brains of diseased mice and, unexpectedly, no increase in immunoreactivity to glial fibrillar acidic protein. The incidence and severity of BDV-induced disease varied dramatically among mouse strains. While only 13% of the infected C57BL/6 mice showed disease symptoms, which were mostly transient, more than 80% of the infected MRL mice developed severe neurological disorder. In spite of these differences in susceptibility to disease, BDV replicated to comparable levels in the brains of mice of the various strains used. Intracerebral infections of newborn β2-microglobulin-deficient C57BL/6 and MRL mice, which both lack CD8⁺ T cells, did not result in meningoencephalitis or neurological disease, indicating that the BDV-induced neurological disorder in mice is a cytotoxic T-cell-mediated immunopathological process. With this new animal model it should now be possible to characterize the disease-inducing immune response to BDV in more detail.     

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.     

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.     

两种博尔纳病病毒株持续感染PC-12细胞模型的建立

目的构建两种博尔纳病病毒株持续感染的PC-12细胞模型,为研究博尔纳病病毒感染致病机制及比较两种病毒株致病特点的差别提供工具。方法将BDV Strain V株和Hu株分别感染PC-12细胞系,并进行传代培养,最后通过Real time FQ RT-PCR、Western blot、间接免疫荧光方法进行病毒核酸和蛋白的检测。结果在培养传代6代后,两种病毒株感染的PC-12细胞均可检测到BDV核酸及蛋白。结论 BDV Strain V株和Hu株均可在PC-12细胞中复制和表达,两种博尔纳病病毒株持续感染PC-12细胞模型构建成功。     

Differential effects of rabies and borna disease viruses on immediate-early- and late-response gene expression in brain tissues.

In situ hybridization and Northern blot analysis were used to examine expression of the immediate-early-response genes (IEGs) egr-1, junB, and c-fos, and the late response gene encoding enkephalin in the brains of rats infected intranasally with Borna disease virus (BDV) or rabies virus. In both Borna disease and rabies virus infections, a dramatic and specific induction of IEGs was detected in particular regions of the hippocampus and the cortex. Increased IEG mRNA expression overlapped with the characteristic expression patterns of BDV RNA and rabies virus RNA, although relative expression levels of viral RNA and IEG mRNA differed, particularly in the hippocampal formation. Furthermore, the temporal relationship between viral RNA synthesis and activation of IEG mRNA expression in BDV infection differed markedly from that in rabies virus infection, suggesting that IEG expression is upregulated by different mechanisms. Expression of proenkephalin (pENK) mRNA was also significantly increased in BDV infection, whereas in rabies virus infection, pENK mRNA levels and also the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were reduced at terminal stages of the disease, probably reflecting a generalized suppression of cellular protein synthesis due to massive production of rabies virus mRNA. The correlation between activated IEG mRNA expression and the strong increase in viral RNA raises the possibility that IEG products induce some phenotypic changes in neurons that render them more susceptible to viral replication.