Persistent Borna Disease Virus Infection Research Articles

The regulation of persistent Borna disease virus infection by RNA silencing factors in human cells

Viral infection induces diverse cellular immune responses. Some viruses induce the production of antiviral cytokines, alterations of endogenous gene expression, and apoptosis; however, other viruses replicate without inducing such responses, enabling them to persistently infect cells. Infection by Borna disease virus type 1 (BoDV-1) can result in fatal immune-mediated encephalitis, including in humans, yet infection of cells in vitro is generally persistent. The regulatory mechanisms underlying this persistent infection remain unclear. Here, we show that an enhancer of RNA-silencing, TRBP, positively regulates BoDV RNA level in human cells. Knockdown of TRBP decreased BoDV RNA levels in persistently-infected cells, whereas overexpression of TRBP increased BoDV RNA levels. To investigate the mechanism underlying this phenomenon, we performed immunoprecipitation assays and found that TRBP interacts with BoDV RNA. Furthermore, we performed cell fractionation, which revealed that persistent infection with BoDV does not alter the localization of TRBP and other RNA silencing factors in cells. Our results showed the regulation of persistent BoDV infection by RNA-silencing factors in human cells.

Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model

Neonatal rat infection with Borna disease virus (BDV), termed neonatal Borna disease, is an established model for investigating the BDV-associated pathogenesis of neurodevelopmental abnormalities. BDV produces a persistent noncytolytic infection in all culture cell systems assayed to date, while persistent infection in neonatal rats results in a progressive loss of hippocampal granule cells, cerebellar Purkinje cells, and cortical GABA-ergic neurons. Persistent infection also results in behavioral deficits including hyperactivity, cognitive impairment, and abnormal social behavior. However, the molecular mechanisms underlying the neuronal degeneration and behavioral abnormalities remain unclear. Using a metabolomic approach based on gas chromatography coupled with mass spectrometry in conjunction with statistical pattern recognition, the metabolic changes in response to BDV Hu-H1 infection were characterized in the rat hippocampus, cerebellum, and cortex. Metabonomic profiling revealed significant perturbations in nucleotide (e.g., adenosine, uracil, inosine, adenosine-5′-monophosphate, uridine-5′-monophosphate, d-ribose 5-phosphate, and sedoheptulose 7-phosphate), amino acid (e.g., lysine, glycine, phenylalanine, tyrosine, proline, serine, cysteine, aspartic acid, pyroglutamic acid, and γ-aminobutyric acid), lipid (e.g., cholesterol, myristic acid, stearic acid, palmitic acid, 1-monopalmitoylglycerol, and arachidonic acid), and energy (e.g., glucose, lactose, 3-phosphoglyceric acid, and pyruvic acid) metabolites. These metabolites participate in pathways crucial to viral proliferation and neurotransmitter homeostasis. This metabolomic profiling study provides insight into the pathogenic mechanisms of BDV and new directions with which to investigate the in vivo effects of persistent BDV infection.

Bornavirus Closely Associates and Segregates with Host Chromosomes to Ensure Persistent Intranuclear Infection

Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.

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.

Loss of connexin36 in rat hippocampus and cerebellar cortex in persistent Borna disease virus infection

Neonatal Borna disease virus (BDV) infection of the Lewis rat leads to progressive degeneration of dentate gyrus granule cells, and cerebellar Purkinje neurons. Our aim here was to clarify whether BDV interfered with the formation of electrical synapses, and we, therefore, analysed expression of the neuronal gap junction protein connexin36 (Cx36) in the Lewis rat hippocampal formation, and cerebellar cortex, 4 and 8 weeks after neonatal infection. Semiquantitative RT-PCR, revealed a BDV-dependent decrease in Cx36 mRNA in the hippocampal formation 4 and 8 weeks post-infection (p.i.), and in the cerebellar cortex 8 weeks p.i. Correspondingly, immunofluorescent staining revealed reduced Cx36 immunoreactivity in both dentate gyrus, and ammons horn CA3 region, 4 and 8 weeks post-infection. In the cerebellar cortex, Cx36 immunoreactivity was detected only 8 weeks post-infection in the molecular layer, where it was down regulated by BDV. Our findings demonstrate, for the first time, distinct BDV-dependent reductions in Cx36 mRNA and protein in the rat hippocampal formation and cerebellar cortex, suggesting altered neuronal network properties to be an important feature of persistent viral brain infections.

Layer specific changes of astroglial gap junctions in the rat cerebellar cortex by persistent Borna Disease Virus infection

Neonatal Borna Disease Virus (BDV) infection of the Lewis rat brain, leads to Purkinje cell degeneration, in association with astroglial activation. Since astroglial gap junctions (GJ) are known to influence neuronal degeneration, we investigated BDV dependent changes in astroglial GJ connexins (Cx) Cx43, and Cx30 in the Lewis rat cerebellum, 4, and 8 weeks after neonatal infection. On the mRNA level, RT-PCR demonstrated a BDV dependent increase in cerebellar Cx43, and a decrease in Cx30, 8, but not 4 weeks p.i. On the protein level, Western blot analysis revealed no overall upregulation of Cx43, but an increase of its phosphorylated forms, 8 weeks p.i. Cx30 protein was downregulated. Immunohistochemistry revealed a BDV dependent reduction of Cx43 in the granular layer (GL), 4 weeks p.i. 8 weeks p.i., Cx43 immunoreactivity recovered in the GL, and was induced in the molecular layer (ML). Cx30 revealed a BDV dependent decrease in the GL, both 4, and 8 weeks p.i. Changes in astroglial Cxs correlated not with expression of the astrogliotic marker GFAP, which was upregulated in radial glia. With regard to functional coupling, primary cerebellar astroglial cultures, revealed a BDV dependent increase of Cx43, and Cx30 immunoreactivity and in spreading of the GJ permeant dye Lucifer Yellow. These results demonstrate a massive, BDV dependent reorganization of astroglial Cx expression, and of functional GJ coupling in the cerebellar cortex, which might be of importance for the BDV dependent neurodegeneration in this brain region.

Persistent Borna Disease Virus infection changes expression and function of astroglial gap junctions in vivo and in vitro

Neonatal Borna Disease Virus (BDV) infection of the Lewis rat brain leads to dentate gyrus (DG) degeneration, underlying mechanisms are not fully understood. Since astroglial gap junction (GJ) coupling is known to influence neurodegenerative processes, the question arose whether persistent BDV infection influences astroglial connexins (Cx) Cx43 and Cx30 in the hippocampal formation (HiF) of Lewis rats. RT-PCR and Western blot analysis of forebrain (FB) samples revealed a virus dependent reduction of both Cx types 8 but not 4 weeks post infection (p.i.). Immunohistochemistry revealed an increase of Cx43 in the DG and a decrease in the CA3 region 4 and 8 weeks p.i. Cx30, which was detectable only 8 weeks p.i., revealed a BDV dependent increase in DG and CA3 regions. BDV dependent astrogliosis as revealed by immunodetection of glial fibrillary acidic protein (GFAP) correlated not with astroglial connexin expression. With regard to functional coupling as revealed by scrape loading, BDV infection resulted in increased spreading of the GJ permeant dye Lucifer yellow in primary hippocampal astroglial cultures, and in increased expression of Cx43 and Cx30 as revealed by immunocytochemistry. In conclusion, persistent BDV infection of the Lewis rat brain leads to changes in astroglial Cx expression both in vivo and in vitro and of functional coupling in vitro. Distribution and time course of these changes suggest them to be a direct result of neurodegeneration in the DG and an indirect effect of neuronal deafferentiation in the CA3 region.

Activation of Microglia by Borna Disease Virus Infection: In Vitro Study

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to the certain neuronal populations. Since persistent BDV infection of neurons in vitro is noncytolytic and noncytopathic, 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 brain have not been investigated. To address these issues, activation of primary rat microglial cells was studied following exposure to purified BDV or to persistently BDV-infected primary cortical neurons or after BDV infection of primary mixed neuron-glial cultures. Neither purified virus nor BDV-infected neurons alone activated primary microglia as assessed by the changes in cell shape or production of the proinflammatory cytokines. In contrast, in the BDV-infected primary mixed cultures, we observed proliferation of microglia cells that acquired the round morphology and expressed major histocompatibility complex molecules of classes I and II. These manifestations of microglia activation were observed in the absence of direct BDV infection of microglia or overt neuronal toxicity. In addition, compared to uninfected mixed cultures, activation of microglia in BDV-infected mixed cultures was associated with a significantly greater lipopolysaccharide-induced release of tumor necrosis factor alpha, interleukin 1beta, and interleukin 10. Taken together, the present data are the first in vitro evidence that persistent BDV infection of neurons and astrocytes rather than direct exposure to the virus or dying neurons is critical for activating microglia.

Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.

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.

Learning deficits in mice with persistent Borna disease virus infection of the CNS associated with elevated chemokine expression

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes a CD8 + T cell-mediated neurological disease in certain mouse strains. We established asymptomatic persistent central nervous system (CNS) infections in mutant C57BL/10J mice that lack functional CD8 + T cells. When analyzed at adult age for spatial learning abilities in a water maze, BDV-infected mice showed slightly impaired escape performance while their exploratory behavior in an openfield test was indistinguishable from uninfected control mice. Histological and molecular biological analysis revealed extensive viral spread throughout the CNS of infected animals. Most neurons of the hippocampus contained viral antigen, but there was no overt loss of neurons from this structure. We found almost unchanged levels of the proinflammatory cytokines IL-1β and TNF-α, but clearly increased levels of the chemokines IP-10 and RANTES in brains of infected mice. Re-examination of water maze data revealed that only infected mice with IP-10 transcript levels above a certain threshold showed impaired performance, whereas the performance of infected mice with lower IP-10 levels was indistinguishable from uninfected controls. This suggests that BDV infection can disturb the function of the mammalian CNS without causing overt neuronal loss, and that the magnitude of virus-induced chemokine production in the CNS correlates with the degree of impairment.

Persistent Borna disease virus infection of neonatal rats causes brain regional changes of mRNAs for cytokines, cytokine receptor components and neuropeptides

Borna disease virus (BDV) replicates in brain cells. The neonatally infected rat with BDV exhibits developmental-neuromorphological abnormalities, neuronal cytolysis, and multiple behavioral and physiological alterations. Here, we report on the levels of interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), IL-1 receptor type I (IL-1RI), IL-1 receptor accessory protein (IL-1R AcP) I and II, glycoprotein 130, and various neuropeptide mRNAs in the cerebellum, parieto-frontal cortex, hippocampus and hypothalamus of BDV-infected rats at 7 and 28 days postintracerebral BDV inoculation. The data show that cytokine and neuropeptide mRNA components are abnormal and differentially modulated in brain regions. IL-1β, TNF-α and TGF-β1 mRNA levels were up-regulated in all brain regions following BDV inoculation. The same cerebellar samples from BDV-infected animals exhibited the highest levels of IL-1β, IL-1Ra, TNF-α, IL-1RI, and IL-1R AcP II mRNA expression. The profiles of IL-1β, IL-1Ra, TNF-α, and TGF-β1 mRNA induction in the cerebellar samples were highly intercorrelated, indicating an association among cytokine ligand mRNAs. Cytokine mRNA induction was differentially up-regulated among brain regions, except for TGF-β1. Specificity of transcriptional changes in response to BDV infection is also suggested by the up-regulation of cytokine and neuropeptide Y mRNAs associated with down-regulation of pro-opiomelanocortin, and with no change of IL-1R AcP I, dynorphin and leptin receptor mRNAs in the same brain region samples. Other data also show a differential mRNA component modulation in distinct brain regions obtained from the same rats depending on the stage of BDV infection. The conclusion of these studies is that cytokines may play a role in the neuropathophysiology of neonatally BDV-infected rats.

Neonatal Borna disease virus infection in the rat causes a loss of Purkinje cells in the cerebellum.

Viral insults that occur during early postnatal periods, can affect neuronal systems which exhibit significant postnatal development, such as the cerebral cortex and cerebellum. Borna disease virus (BDV) is a single-strand RNA virus which replicates in the nervous system of many species after experimental inoculation and causes acute neurological disease. Neonatal rats infected with BDV do not mount an aggressive response to the virus like their adult counterparts, but instead develop a persistent BDV infection with less overt clinical sequelae. Recently, the cerebellum, a neural structure associated with regulation of motor behavior, and perhaps with higher cognitive functions, has been demonstrated to be a target of neonatal BDV infections in rats (Bautista et al, 1995). In the present study neonatal rats were infected with BDV and their cerebella were analyzed histologically and immunohistochemically at 7 months of age. The cerebella of infected animals were reduced in size but normal foliation and laminar organization was present. However, as visualized with immunohistochemistry for the Purkinje cell-specific antigen calbindin, there were numerous gaps within the Purkinje cell layer and in the molecular layer which contains the Purkinje cell dendritic trees. We estimated the number of Purkinje cells and found there was an approximately 75% loss of PC in adult rats neonatally infected with BDV. These results suggest that neonatal BDV infection may either (1) target the PC and cause the death of these cells directly or (2) acts indirectly by triggering an immune response which is then responsible for the loss of these cells.

Virus-specific CD4+ T cells eliminate borna disease virus from the brain via induction of cytotoxic CD8+ T cells.

Persistent Borna disease virus infection of the brain can be prevented by treatment of naive rats with a virus-specific CD4+ T-cell line prior to infection. In rats receiving this treatment, only a transient low-level encephalitis was seen compared to an increasingly inflammatory reaction in untreated infected control rats. Virus replication was found in the brain for several days after infection before the virus was cleared from the central nervous system. The loss of infectivity from the brain was confirmed by negative results by reverse transcription-PCR with primers for mRNA, by in situ hybridization for both genomic and mRNA, and by immunohistology. Most importantly, in vitro assays revealed that the T-cell line used for transfusion had no cytotoxic capacity. The kinetics of virus clearance were paralleled by the appearance of CD8+ T cells and the expression of perforin in the brain. Testing of lymphocytes isolated from the brains of CD4+ T-cell-treated rats after challenge revealed high cytotoxic activity due to the presence of CD8+ cytotoxic T cells at time points when brain lymphocytes from infected control rats induced low-level cytolysis of target cells. Neutralizing antiviral antibodies and gamma interferon were shown not to be involved in the elimination of virus from the brain.

Developmental injury to the cerebellum following perinatal Borna disease virus infection

In rats infected as neonates, Borna disease virus (BDV) infection causes neuroanatomical, behavioral and physiological abnormalities without encephalitis. Neonatal infection with BDV provides a powerful model for studying the effects of virus replication on brain development without inflammation-induced brain damage. Here we report that neonatal BDV infection interfered with cerebellar development in the Lewis rat. Based on cerebellar cross-sectional area measurements, abnormal cerebellar growth was first noted between 7 and 14 days after infection. Reactive astrocytosis was evident by three days after infection, even without encephalitis, and even before identification of viral proteins in the cerebellum. While neonatal BDV infection caused a significant loss in granule cells, infected granule cells were not identified. BDV proteins were readily detected in the Purkinje cells. Thus, persistent BDV infection of Purkinje cells, but not granule cells, was associated with loss of granule cells during cerebellar development, in the absence of encephalitis.