Borna disease virus replication in organotypic hippocampal slice cultures from rats results in selective damage of dentate granule cells

In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a post-maturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.     

Measles virus infection induces chemokine synthesis by neurons

The role that neurons play in the induction of the immune response following CNS viral infection is poorly understood, largely owing to the belief that these cells are immunologically quiescent. In this report, we show that virus infection of neurons results in the synthesis of proinflammatory chemokines, which are early and important mediators of leukocyte recruitment to sites of viral infection. For these studies, a transgenic mouse model of neuron-restricted measles virus (MV) infection was used. Inoculation of immunocompetent and immunodeficient transgenic adult mice resulted in CNS induction of the mRNAs encoding IFN-gamma inducible protein of 10 kD, monokine inducible by gamma and RANTES. Colocalization of chemokine proteins with MV-infected neurons was detected by immunofluorescence in infected brain sections. Both IFN-gamma inducible protein 10 kD and RANTES were also induced in MV-infected primary hippocampal neurons cultured from transgenic embryos, as shown by RNase protection assay, confocal microscopy, and ELISA. Interestingly, neuronal infection with another RNA virus (lymphocytic choriomeningitis virus) was not associated with induction of these chemokines. In immunocompetent mice, chemokine synthesis preceded the infiltration of T lymphocytes, and chemokine ablation by neutralizing Abs resulted in a 20-50% reduction in the number of infiltrating lymphocytes. Collectively, these data indicate that neurons play an important role in the recruitment of a protective antiviral response to the CNS following viral infection, although such a role may be virus type-dependent.     

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.     

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.     

Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease

The nucleoprotein (N) of Borna disease virus (BDV) is the major target of the disease-inducing antiviral CD8 T-cell response in the central nervous system of mice. We established two transgenic mouse lines which express BDV-N in either neurons (Neuro-N) or astrocytes (Astro-N). Despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. When Neuro-N mice were infected as adults, replication of BDV was severely impaired and was restricted to brain areas with a low density of transgene-expressing cells. Notably, the virus failed to replicate in the transgene-expressing granular and pyramidal neurons of the hippocampus (which are usually the preferred host cells of BDV). When Neuro-N mice were infected within the first 5 days of life, replication of BDV was not suppressed in most neurons, presumably because the onset of transgene expression in the brain occurred after these cells became infected with BDV. Astro-N mice remained susceptible to BDV infection, but they were resistant to BDV-induced neurological disorder. Unlike their nontransgenic littermates, Neuro-N mice with persistent BDV infection did not develop neurological disease after immunization with a vaccinia virus vector expressing BDV-N. In contrast to the situation in wild-type mice, this treatment also failed to induce N-specific CD8 T cells in the spleens of both transgenic mouse lines. Thus, while resistance to BDV infection in N-expressing neurons appeared to result from untimely expression of a viral nucleocapsid component, the resistance to BDV-induced neuropathology probably resulted from immunological tolerance.     

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.     

Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-gamma) secretion by CD8(+) T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8(+) T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-gamma-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4(+) T cells and localized to similar areas. Although CD8(+) T cells from all three donors suppressed virus replication in the CNS, GKO CD8(+) T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8(+) T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-gamma suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-gamma but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8(+) T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8(+) T cells in the absence of CD4(+) T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.     

Perforin-mediated effector function within the central nervous system requires IFN-gamma-mediated MHC up-regulation

CD8(+) T cells infiltrating the CNS control infection by the neurotropic JHM strain of mouse hepatitis virus. Differential susceptibility of infected cell types to clearance by perforin or IFN-gamma uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8(+) T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN-gamma (PKO/GKO) or selectively reconstituted for each function by transfer of CD8(+) T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8(+) T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4(+) T cells in CNS infiltrates. CD8(+) T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN-gamma-deficient donor CD8(+) T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN-gamma coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN-gamma within the CNS but also demonstrate IFN-gamma-dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN-gamma plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8(+) T cell effector function, and demyelination.     

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.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

Type I interferons are essential in controlling neurotropic coronavirus infection irrespective of functional CD8 T cells

Neurotropic coronavirus infection induces expression of both beta interferon (IFN-beta) RNA and protein in the infected rodent central nervous system (CNS). However, the relative contributions of type I IFN (IFN-I) to direct, cell-type-specific virus control or CD8 T-cell-mediated effectors in the CNS are unclear. IFN-I receptor-deficient (IFNAR(-/-)) mice infected with a sublethal and demyelinating neurotropic virus variant and those infected with a nonpathogenic neurotropic virus variant both succumbed to infection within 9 days. Compared to wild-type (wt) mice, replication was prominently increased in all glial cell types and spread to neurons, demonstrating expanded cell tropism. Furthermore, increased pathogenesis was associated with significantly enhanced accumulation of neutrophils, tumor necrosis factor alpha, interleukin-6, chemokine (C-C motif) ligand 2, and IFN-gamma within the CNS. The absence of IFN-I signaling did not impair induction or recruitment of virus-specific CD8 T cells, the primary adaptive mediators of virus clearance in wt mice. Despite similar IFN-gamma-mediated major histocompatibility complex class II upregulation on microglia in infected IFNAR(-/-) mice, class I expression was reduced compared to that on microglia in wt mice, suggesting a synergistic role of IFN-I and IFN-gamma in optimizing class I antigen presentation. These data demonstrate a critical direct antiviral role of IFN-I in controlling virus dissemination within the CNS, even in the presence of potent cellular immune responses. By limiting early viral replication and tropism, IFN-I controls the balance of viral replication and immune control in favor of CD8 T-cell-mediated protective functions.     

High-magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice

The neurotropic JHM strain of mouse hepatitis virus (MHV) causes acute encephalitis and chronic demyelinating encephalomyelitis in rodents. Previous results indicated that CD8 T cells infiltrating the central nervous system (CNS) were largely antigen specific in both diseases. Herein we show that by 7 days postinoculation, nearly 30% of the CD4 T cells in the acutely infected CNS were MHV specific by using intracellular gamma interferon (IFN-gamma) staining assays. In mice with chronic demyelination, 10 to 15% of the CD4 T cells secreted IFN-gamma in response to MHV-specific peptides. Thus, these results show that infection of the CNS is characterized by a large influx of CD4 T cells specific for MHV and that these cells remain functional, as measured by cytokine secretion, in mice with chronic demyelination.     

Interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of CXCL10/IP-10 gene expression by astrocytes in vivo and in vitro.

The CXC chemokine gamma interferon (IFN-gamma)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3(+) T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.     

Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells

The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.     

Borna disease virus glycoprotein is required for viral dissemination in neurons

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.     

Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.     

Contributions of CD8+ T cells and viral spread to demyelinating disease

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.