Nucleoprotein (P40) Binding to 5HT2C Receptors (5HT2CR) is the Key Point in the Pathogenesis of BoDV-1-Infected Hosts.

Nucleoprotein (P40) is one of the most important proteins of Borna disease virus 1 (BoDV-1), but which proteins it would bind to in the pathogenesis of BoDV-1-infected hosts is unknown. We used lentivirus LV5-P40 overexpressing P40 to infect primary hippocampal neurons and characterized the interactome of P40 with co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS) analysis. These interacting protein partners revealed the pathogenesis of BoDV-1-infected hosts. We also show for the first time that P40 interacts with 5HT2CR in rat neurons, which may be the molecular basis leading to neuropsychiatric diseases such as anxiety disorders and behavioral abnormalities after BoDV-1 infection of hosts.

Intranasal Borna Disease Virus (BoDV-1) Infection: Insights into Initial Steps and Potential Contagiosity.

Mammalian Bornavirus (BoDV-1) typically causes a fatal neurologic disorder in horses and sheep, and was recently shown to cause fatal encephalitis in humans with and without transplant reception. It has been suggested that BoDV-1 enters the central nervous system (CNS) via the olfactory pathway. However, (I) susceptible cell types that replicate the virus for successful spread, and (II) the role of olfactory ensheathing cells (OECs), remained unclear. To address this, we studied the intranasal infection of adult rats with BoDV-1 in vivo and in vitro, using olfactory mucosal (OM) cell cultures and the cultures of purified OECs. Strikingly, in vitro and in vivo, viral antigen and mRNA were present from four days post infection (dpi) onwards in the olfactory receptor neurons (ORNs), but also in all other cell types of the OM, and constantly in the OECs. In contrast, in vivo, BoDV-1 genomic RNA was only detectable in adult and juvenile ORNs, nerve fibers, and in OECs from 7 dpi on. In vitro, the rate of infection of OECs was significantly higher than that of the OM cells, pointing to a crucial role of OECs for infection via the olfactory pathway. Thus, this study provides important insights into the transmission of neurotropic viral infections with a zoonotic potential.

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell.

Borna disease virus (BDV) is a non-segmented negative-stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell-cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell-cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin-mediated processing of GP and demonstrate that cleaved and fusion-active GP is strictly necessary for the cell-to-cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus-glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.

Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes.

Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses.

Neuropathogenesis of persistent infection with Borna disease virus.

Borna disease virus (BDV), belonging to the non-segmented, negative-stranded RNA viruses, persistently infects the central nervous system of many mammals. Neonatal BDV infection in rodent models induces neurodevelopmental disturbance without overt inflammatory responses, resulting in a wide range of neurobehavioral abnormalities, such as anxiety, abnormal play behaviors, and cognitive deficits, resembling those of autism patients. Therefore, studies of BDV could provide a valuable model to investigate neuropathogenesis of neurodevelopmental disorders. However, the detailed neuropathogenesis of BDV has not been revealed. Here, we proposed two novel mechanisms that may contribute to BDV neuropathology. The first mechanism is abnormal IGF signaling. Using transgenic mice expressing BDV P protein in glial cells (P-Tg) that show neurobehavioral abnormalities resembling those in BDV-infected animals, we found that the upregulation of insulin-like growth factor (IGF) binding protein 3 in the astrocytes disturbs the IGF signaling and induces the Purkinje cell loss in BDV infection. The other is the integration of BDV sequences into the host genome. We recently found that BDV mRNAs are reverse-transcribed and integrated into the genome of infected cells. BDV integrants have the potential to produce their translated products or piRNAs, suggesting that BDV might exhibit the pathogenicity thorough these molecules. We also demonstrated that BDV integrants affect neighboring gene expression. Collectively, BDV integrants may alter transcriptome of infected cells, affecting BDV neuropathology.

Analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein.

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.

Epidemiology and host spectrum of Borna disease virus infections.

Borna disease virus (BDV) has gained lot of interest because of its zoonotic potential, ability to introduce cDNA of its RNA transcripts into host genomes, and ability to cause severe neurobehavioural diseases. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep, known in central Europe for centuries. According to current knowledge, BDV or a close relative also infects several other species, including humans at least occasionally, in central Europe and elsewhere, but the existence of potential 'human Borna disease' with its suspected neuropsychiatric symptoms is highly controversial. The recent detection of endogenized BDV-like genes in primate and various other vertebrate genomes confirms that at least ancient bornaviruses did infect our ancestors. The epidemiology of BDV is largely unknown, but accumulating evidence indicates vectors and reservoirs among small wild mammals. The aim of this review is to bring together the current knowledge on epidemiology of BDV infections. Specifically, geographical and host distribution are addressed and assessed in the critical light of the detection methods used. We also review some salient clinical aspects.

Upregulation of insulin-like growth factor binding protein 3 in astrocytes of transgenic mice that express Borna disease virus phosphoprotein.

In a previous study, we demonstrated that transgenic mice that express Borna disease virus (BDV) phosphoprotein (P) in astrocytes show striking neurobehavioral abnormalities resembling those in BDV-infected animals. To understand the molecular disturbances induced by the expression of P in astrocytes, we performed microarray analysis with cultured astroglial cells transiently expressing P. We showed that expression of insulin-like growth factor binding protein 3 mRNA increases not only in P-expressing cultured cells but also in astrocytes from the cerebella of P transgenic mice (P-Tg). Furthermore, we demonstrated that insulin-like growth factor signaling is disturbed in the P-Tg cerebellum, a factor that might be involved in the increased vulnerability of Purkinje cell neurons in the brain.

Avian bornavirus associated with fatal disease in psittacine birds.

Thanks to new technologies which enable rapid and unbiased screening for viral nucleic acids in clinical specimens, an impressive number of previously unknown viruses have recently been discovered. Two research groups independently identified a novel negative-strand RNA virus, now designated avian bornavirus (ABV), in parrots with proventricular dilatation disease (PDD), a severe lymphoplasmacytic ganglioneuritis of the gastrointestinal tract of psittacine birds that is frequently accompanied by encephalomyelitis. Since its discovery, ABV has been detected worldwide in many captive parrots and in one canary with PDD. ABV induced a PDD-like disease in experimentally infected cockatiels, strongly suggesting that ABV is highly pathogenic in psittacine birds. Until the discovery of ABV, the Bornaviridae family consisted of a single species, classical Borna disease virus (BDV), which is the causative agent of a progressive neurological disorder that affects primarily horses, sheep, and some other farm animals in central Europe. Although ABV and BDV share many biological features, there exist several interesting differences, which are discussed in this review.

Visualizing viral dissemination in the mouse nervous system, using a green fluorescent protein-expressing Borna disease virus vector.

Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.

Cell entry of Borna disease virus follows a clathrin-mediated endocytosis pathway that requires Rab5 and microtubules.

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.

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.

Reverse genetics approaches of Borna disease virus: applications in development of viral vectors and preventive vaccines.

The plasmid-based reverse genetics system, which involves generation of recombinant viruses from cloned cDNA, has accelerated the understanding of clinical and virological aspects of different viruses. Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that causes persistent intranuclear infection in various vertebrate species. Since its first report, reverse genetics approaches with modified strategies have greatly improved rescue efficiency of recombinant BoDV and enhanced the understanding of function of each viral protein and mechanism of intranuclear persistency. Here, we summarize different reverse genetics approaches of BoDV and recent developments in the use of reverse genetics for generation of viral vectors for gene therapy and virus-like particles for potential preventive vaccines.

Binding properties of GP1 protein of Borna disease virus.

The surface glycoprotein (G) of Borna disease virus (BDV) plays central roles in the process of viral entry. BDV G is cleaved by cellular furin-like proteases into two components, GP1 and GP2. Although GP1 is involved in the virus entry into cells, the binding activity of GP1 to cells is unknown. Therefore, we expressed the wild-type GP1 and a variety of GP1 deletion mutants that were FLAG-tagged at the C-terminus in human embryonic kidney 293T cells. These proteins were then purified using an anti-FLAG antibody and evaluated for their ability to bind to cell lines. GP1 bound to BDV-permissive cells but not to non-permissive cells. GP1 also inhibited BDV infection via its binding to cells. This binding assay should prove useful to map the receptor-binding domain of BDV.

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.

Infection of the enteric nervous system by Borna disease virus (BDV) upregulates expression of Calbindin D-28k.

Borna disease virus (BDV) is a neurotropic agent infecting distinct neuronal subpopulations in the central nervous system of various mammalian species possibly including humans. Horses, a major natural host for BDV, show gastrointestinal dysfunctions besides characteristic neurological symptoms. Therefore, we hypothesized that enteric neurons may be targets of BDV replication. The presence of BDV-specific antigen in subpopulations of the ENS was investigated. Four-week-old Lewis rats were infected intracerebrally and sacrificed 4-14 weeks post infection (p.i.). BDV-immunoreactive neurons were found in submucous and myenteric neurons of the proximal colon. Fourteen weeks p.i., the proportion of BDV-positive neurons was 44+/-17 and 24+/-7% in the submucous and myenteric plexus, respectively. The majority of BDV-positive myenteric neurons showed immunoreactivity for choline acetyltransferase. Expression of Calbindin D-28k (CALB) was found in 96% of submucous and 67% of myenteric BDV-immunoreactive neurons. Additionally, the number of CALB-immunoreactive neurons was significantly higher in the myenteric plexus of infected rats compared to controls. These data indicate that BDV infects specific subpopulations of enteric neurons. Therefore, the ENS might serve as a site for BDV replication and as an immunoprivileged reservoir for BDV. In addition, upregulation of CALB in neurons of the myenteric plexus is probably induced during BDV-infection.

The association between Borna Disease Virus and schizophrenia: A systematic review and meta-analysis.

INTRODUCTION: Schizophrenia is a disabling psychiatric disorder. The role of Borna Disease Virus (BDV) in the etiology of schizophrenia has been suggested by several studies. However, the existence of such association remained controversial. The present meta-analysis was conducted to evaluate this association. METHOD: This systematic review and meta-analysis was conducted using preferred reporting items for systematic reviews and meta-analysis (PRISMA). Online databases including Scopus, PubMed, Science direct, Embase, PsycINFO, Web of Science and Google scholar search engine were searched until January 15, 2017. The heterogeneity of the studies was evaluated using Cochran's Q test and I2 statistic. Finally, random effects model was used for combining the results using Stata software version 11.1. RESULT: Overall, 30 studies containing 2533 cases and 4004 controls were included in the meta-analysis. The combined odds ratio (OR) for the relationship between BDV and schizophrenia was estimated to be 2.72 (95%CI: 1.75-4.20). This association based on RT-PCR, WB, IFA, EIA, RLA, ECLIA methods was estimated to be 3.83 (95%CI: 1.59-9.20), 4.99 (95%CI: 1.80-13.85), 1.27 (95%CI: 0.23-7.12), 2.26 (95%CI: 0.48-10.64), 1.67 (95%CI: 0.50-5.56) and 2.88 (95%CI: 1.38-6.01), respectively. Subgroup analysis according to WBC, serum and plasma samples was estimated to be 3.31 (95%CI: 1.19-9.25), 2.21 (95% CI: 1.17-4.17), 2.21 (95%CI: 1.03-4.73) and 7.89 (95%CI: 1.75-35.53), respectively. CONCLUSION: The results indicated the role of BDV in the etiology of schizophrenia.

Characterization of a Borna disease virus field isolate which shows efficient viral propagation and transmissibility.

To investigate the biological characteristics of field isolates of Borna disease virus (BDV), as well as to understand BDV infections outside endemic countries, we isolated the virus from brain samples of a heifer with Borna disease in Japan. We demonstrate that the brain lysate contained replication products of BDV and induced viral propagation in rat glioma cells, suggesting that a replication-competent BDV existed in the bovine brain. This field strain of BDV, named Bo/04w, showed efficient viral release and transmissibility and also displayed a distinct pattern of expression of viral phosphoprotein (P) during infection, as compared with laboratory-adapted BDV strains. Interestingly, we found the level of P to be significantly low in cells infected with Bo/04w, and the transcription of this isolate to be more efficient than that of laboratory strain of BDV. These results indicated that the field isolate may regulate the expression of P at an optimal level in infected cells. We also confirmed that Bo/04w maintains biological significance in neonatal gerbil brain. Sequencing revealed that despite the biological differences, the field isolate is closely related genetically to the laboratory strains of BDV. We discuss here the sequence similarities between BDV isolates from endemic and nonendemic countries.

Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers.

Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat.

Viruses as an etiology of obesity.

Obesity is a serious chronic disease that has numerous etiologies. The prevalence of obesity has increased dramatically since about 1980 in the United States and worldwide in both developed and developing countries. This rapid spread is compatible with an infectious origin. This review discusses the 5 animal viruses and 3 human viruses that have been shown to cause obesity and examines the evidence to date for virus-induced obesity. The obesogenic animal viruses include canine distemper virus, Rous-associated virus type 7, Borna disease virus, scrapie agent, and SMAM-1. The first 4 viruses attack the central nervous system to produce obesity. SMAM-1, an avian adenovirus from India, acts directly on adipocytes and is the only animal virus that is associated with human obesity. The 3 human adenoviruses, adenovirus (Ad) 36, Ad-37, and Ad-5, that are associated with obesity also affect adipocytes directly. These viruses stimulate enzymes and transcription factors that cause accumulation of triglycerides and differentiation of preadipocytes into mature adipocytes. Ad-5 and Ad-37 have been shown to cause obesity in animals. Ad-36 has been studied the most and is the only human adenovirus to date that has been linked with human obesity. Ad-36 causes obesity in chickens, mice, rats, and monkeys and was present in 30% of obese humans and 11% of nonobese humans. In twins discordant for infection with Ad-36, the infected twins were heavier and fatter than their cotwins. The growing body of evidence demonstrating that viruses produce human obesity supports the concept that at least some of the worldwide epidemic of obesity in the past 25 years is due to viral infections.