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Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease, a fatal neurologic disorder of domestic mammals and humans, resulting from spill-over infection from its natural reservoir host, the bicolored white-toothed shrew (Crocidura leucodon). The known BoDV-1-endemic area is remarkably restricted to parts of Germany, Austria, Switzerland and Liechtenstein. To gain comprehensive data on its occurrence, we analysed diagnostic material from suspected BoDV-1-induced encephalitis cases based on clinical and/or histopathological diagnosis. BoDV-1 infection was confirmed by RT-qPCR in 207 domestic mammals, 28 humans and seven wild shrews. Thereby, this study markedly raises the number of published laboratory-confirmed human BoDV-1 infections and provides a first comprehensive summary. Generation of 136 new BoDV-1 genome sequences from animals and humans facilitated an in-depth phylogeographic analysis, allowing for the definition of risk areas for zoonotic BoDV-1 transmission and facilitating the assessment of geographical infection sources. Consistent with the low mobility of its reservoir host, BoDV-1 sequences showed a remarkable geographic association, with individual phylogenetic clades occupying distinct areas. The closest genetic relatives of most human-derived BoDV-1 sequences were located at distances of less than 40 km, indicating that spill-over transmission from the natural reservoir usually occurs in the patient´s home region., (© 2024. The Author(s).)

Background: Zoonotic Borna disease virus 1 (BoDV-1) causes fatal encephalitis in humans and animals. Subsequent to the detection of two paediatric cases in a Bavarian municipality in Germany within three years, we conducted an interdisciplinary One Health investigation. We aimed to explore seroprevalence in a local human population with a risk for BoDV-1 exposure as well as viral presence in environmental samples from local sites and BoDV-1 prevalence within the local small mammal population and its natural reservoir, the bicoloured white-toothed shrew (Crocidura leucodon)., Methods: The municipality's adult residents participated in an anonymised sero-epidemiological study. Potential risk factors and clinical symptoms were assessed by an electronic questionnaire. Small mammals, environmental samples and ticks from the municipality were tested for BoDV-1-RNA. Shrew-derived BoDV-1-sequences together with sequences of the two human cases were phylogenetically analysed., Results: In total, 679 citizens participated (response: 41 %), of whom 38 % reported shrews in their living environment and 19 % direct shrew contact. No anti-BoDV-1 antibodies were detected in human samples. BoDV-1-RNA was also undetectable in 38 environmental samples and 336 ticks. Of 220 collected shrews, twelve of 40 C. leucodon (30%) tested BoDV-1-RNA-positive. BoDV-1-sequences from the previously diagnosed two paediatric patients belonged to two different subclades, that were also present in shrews from the municipality., Interpretation: Our data support the interpretation that human BoDV-1 infections are rare even in endemic areas and primarily manifest as severe encephalitis. Sequence analysis linked both previous paediatric human infections to the local shrew population, but indicated independent infection sources., Funding: The project was partly financed by funds of the German Federal Ministry of Education and Research (grant numbers: 01KI2005A, 01KI2005C, 01KI1722A, 01KI1722C, 01KI2002 to MaBe, DR, RGU, DT, BS) as well as by the ReForM-A programme of the University Hospital Regensburg (to MaBa) and by funds of the Bavarian State Ministry of Health, Care and Prevention, project "Zoonotic Bornavirus Focal Point Bavaria - ZooBoFo" (to MaBa, MaBe, BS, MMB, DR, PS, RGU)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.

Competing Interests: Declaration of interests We declare no competing interests.

Background: Borna disease virus 1 (BoDV-1) causes rare human infections within endemic regions in southern and eastern Germany. The infections reported to date have been linked to severe courses of encephalitis with high mortality and mostly irreversible symptoms. Whether BoDV-1 could act as a trigger for other neurological conditions, is, however, incompletely understood., Objectives and Methods: In this study, we addressed the question of whether the presentation of a clinically isolated syndrome (CIS) or of multiple sclerosis (MS) might be associated with a milder course of BoDV-1 infections. Serum samples of 100 patients with CIS or MS diagnosed at a tertiary neurological care center within an endemic region in southern Germany and of 50 control patients suffering from headache were retrospectively tested for BoDV-1 infections., Results: In none of the tested sera, confirmed positive results of anti-BoDV-1-IgG antibodies were retrieved. Our results support the conclusion that human BoDV-1 infections primarily lead to severe encephalitis with high mortality., (© 2023. The Author(s).)

Purpose: Human Borna disease virus (BoDV-1) encephalitis is an emerging disease in Germany. This study investigates the spectrum of human BoDV-1 infection, characterizes anti-BoDV-1-antibodies and kinetics, and compares laboratory test performances., Methods: Three hundred four encephalitis cases, 308 nation-wide neuropsychiatric conditions, 127 well-defined psychiatric cases from Borna disease-endemic areas, and 20 persons with contact to BoDV-1 encephalitis patients or animals were tested for BoDV-1 infections by serology and PCR., Results: BoDV-1 infections were only found in encephalitis patients with residence in, or recent travel to, virus-endemic areas. Antibodies were detected as early as 12 days after symptom onset. Serum antibody levels correlated with disease duration. Serology was ordered after 50% of the disease duration had elapsed, reflecting low awareness. BoDV-1-antibodies were of IgG1 subclass, and the epitope on BoDV-1 antigens was determined. Specificity of the indirect immunofluorescence antibody test (IFAT) and lineblot (LB) from serum and cerebrospinal fluid (CSF), as well as PCR testing from CSF, was 100%. Sensitivity, depending on first or all samples, reached 75-86% in serum and 92-94% in CSF for the IFAT, and 33-57% in serum and 18-24% in CSF for the LB. Sensitivity for PCR in CSF was 25-67%. Positive predictive values were 100% each, while negative predictive values were 99% (IFAT), 91-97% (LB), and 90% (PCR)., Conclusions: There is no hint that BoDV-1 causes other diseases than encephalitis in humans. Awareness has to be increased in virus-endemic areas. Tests are robust but lack sensitivity. Detection of IgG1 against specific peptides may facilitate diagnosis. Screening of healthy individuals is likely not beneficial., (© 2023. The Author(s).)

Borna disease virus (BoDV-1) is a bornavirus prototype that infects the central nervous system of various animal species and can cause fatal encephalitis in various animals including humans. Among the reported anti-BoDV-1 treatments, favipiravir (T-705) is one of the best candidates since it has been shown to be effective in reducing various bornavirus titers in cell culture. However, T-705 effectiveness on BoDV-1 is cell type-dependent, and the molecular mechanisms that explain this cell type-dependent difference remain unknown. In this study, we noticed a fact that T-705 efficiently suppressed BoDV-1 in infected 293T cells, but not in infected SH-SY5Y cells, and sought to identify protein(s) responsible for this cell-type-dependent difference in T-705 efficacy. By comparing the transcriptomes of BoDV-1-infected 293T and SH-SY5Y cells, we identified heart- and neural crest derivatives-expressed protein 2 (HAND2) as a candidate involved in T-705 interference. HAND2 overexpression partly attenuated the inhibitory effect of T-705, whereas HAND2 knockdown enhanced this effect. We also demonstrated an interaction between T-705 and HAND2. Furthermore, T-705 impaired HAND2-mediated host gene expression. Because HAND2 is an essential transcriptional regulator of embryogenesis, T-705 may exhibit its adverse effects such as teratogenicity and embryotoxicity through the impairment of HAND2 function. This study provides novel insights into the molecular mechanisms underlying T-705 interference in some cell types and inspires the development of improved T-705 derivatives for the treatment of RNA viruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Borna disease is a progressive meningoencephalitis caused by spillover of the Borna disease virus 1 (BoDV-1) to horses and sheep and has gained attention due to its zoonotic potential. New World camelids are also highly susceptible to the disease; however, a comprehensive description of the pathological lesions and viral distribution is lacking for these hosts. Here, the authors describe the distribution and severity of inflammatory lesions in alpacas ( n = 6) naturally affected by this disease in comparison to horses ( n = 8) as known spillover hosts. In addition, the tissue and cellular distribution of the BoDV-1 was determined via immunohistochemistry and immunofluorescence. A predominant lymphocytic meningoencephalitis was diagnosed in all animals with differences regarding the severity of lesions. Alpacas and horses with a shorter disease duration showed more prominent lesions in the cerebrum and at the transition of the nervous to the glandular part of the pituitary gland, as compared to animals with longer disease progression. In both species, viral antigen was almost exclusively restricted to cells of the central and peripheral nervous systems, with the notable exception of virus-infected glandular cells of the Pars intermedia of the pituitary gland. Alpacas likely represent dead-end hosts similar to horses and other spillover hosts of BoDV-1., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In 2018, Borna Disease Virus 1 (BoDV-1) was confirmed as a human zoonotic pathogen causing rare but fatal encephalitis in Germany. While diagnostic procedures and the clinical picture have been described, epidemiology remains mysterious. Though endemic areas and a natural reservoir host have been identified with the shrew Crocidura leucodon shedding virus in secretions, transmission events, routes and risk factors are unclear. We performed the first comprehensive epidemiological study, combining a large case series with the first case-control study: We interviewed family members of 20 PCR-confirmed BoDV-1 encephalitis cases deceased in 1996-2021 with a standardized questionnaire covering medical history, housing environment, profession, animal contacts, outdoor activities, travel, and nutrition. Cases' median age was 51 (range 11-79) years, 12/20 were female, and 18/20 lived in the federal state of Bavaria in Southeastern Germany. None had a known relevant pre-existing medical condition. None of the interviews yielded a transmission event such as direct shrew contact, but peridomestic shrew presence was confirmed in 13 cases supporting environmental transmission. Residency in rural areas endemic for animal BoDV-1 was the common denominator of all cases. A subsequent individually matched case-control study revealed residence close to nature in a stand-alone location or on the fringe of the settlement as a risk factor for disease in multivariable analysis with an adjusted OR of 10.8 (95% CI 1.3-89.0). Other variables including keeping cats were not associated with disease. Targeted prevention, future post-exposure-prophylaxis, and timely diagnosis remain challenging.

Human bornavirus encephalitis is an emerging disease caused by the variegated squirrel bornavirus 1 (VSBV-1) and the Borna disease virus 1 (BoDV-1). While characteristic brain magnetic resonance imaging (MRI) changes have been described for BoDV-1 encephalitis, only scarce diagnostic data in VSBV-1 encephalitis exist. We systematically analysed brain MRI scans from all known VSBV-1 encephalitis patients. Initial and follow-up scans demonstrated characteristic T2 hyperintense lesions in the limbic system and the basal ganglia, followed by the brainstem. No involvement of the cerebellar cortex was seen. Deep white matter affection occurred in a later stage of the disease. Strict symmetry of pathologic changes was seen in 62%. T2 hyperintense areas were often associated with low T1 signal intensity and with mass effect. Sinusitis in three patients on the first MRI and an early involvement of the limbic system suggest an olfactory route of VSBV-1 entry. The viral spread could occur per continuitatem to adjacent anatomical brain regions or along specific neural tracts to more distant brain regions. The number and extent of lesions did not correlate with the length of patients' survivals. The overall pattern closely resembles that described for BoDV-1 encephalitis. The exact bornavirus species can thus not be deduced from imaging results alone, and molecular testing and serology should be performed to confirm the causative bornavirus. As VSBV-1 is likely of tropical origin, and MRI investigations are increasingly available globally, imaging techniques might be helpful to facilitate an early presumptive diagnosis of VSBV-1 encephalitis when molecular and/or serological testing is not available.

Background: The Borna disease virus (BoDV-1) is an emerging zoonotic virus causing severe and mostly fatal encephalitis in humans., Methods and Results: A local cluster of fatal BoDV-1 encephalitis cases was detected in the same village three years apart affecting two children. While the first case was diagnosed late in the course of disease, a very early diagnosis and treatment attempt facilitated by heightened awareness was achieved in the second case. Therapy started as early as day 12 of disease. Antiviral therapy encompassed favipiravir and ribavirin, and, after bioinformatic modelling, also remdesivir. As the disease is immunopathogenetically mediated, an intensified anti-inflammatory therapy was administered. Following initial impressive clinical improvement, the course was also fatal, although clearly prolonged. Viral RNA was detected by qPCR in tear fluid and saliva, constituting a possible transmission risk for health care professionals. Highest viral loads were found post mortem in the olfactory nerve and the limbic system, possibly reflecting the portal of entry for BoDV-1. Whole exome sequencing in both patients yielded no hint for underlying immunodeficiency. Full virus genomes belonging to the same cluster were obtained in both cases by next-generation sequencing. Sequences were not identical, indicating viral diversity in natural reservoirs. Specific transmission events or a common source of infection were not found by structured interviews. Patients lived 750m apart from each other and on the fringe of the settlement, a recently shown relevant risk factor., Conclusion: Our report highlights the urgent necessity of effective treatment strategies, heightened awareness and early diagnosis. Gaps of knowledge regarding risk factors, transmission events, and tailored prevention methods become apparent. Whether this case cluster reflects endemicity or a geographical hot spot needs further investigation., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Borna disease virus (BoDV-1) is a highly neurotropic RNA virus that causes neurobehavioral disturbances such as abnormal social activities and memory impairment. Although impairments in the neural circuits caused by BoDV-1 infection induce these disturbances, the molecular basis remains unclear. Furthermore, it is unknown whether anti-BoDV-1 treatments can attenuate BoDV-1-mediated transcriptomic changes in neuronal cells. In this study, we investigated the effects of BoDV-1 infection on neuronal differentiation and the transcriptome of differentiated neuronal cells using persistently BoDV-1-infected cells. Although BoDV-1 infection did not have a detectable effect on intracellular neuronal differentiation processes, differentiated neuronal cells exhibited transcriptomic changes in differentiation-related genes. Some of these transcriptomic changes, such as the decrease in the expression of apoptosis-related genes, were recovered by anti-BoDV-1 treatment, while alterations in the expression of other genes remained after treatment. We further demonstrated that a decrease in cell viability induced by differentiation processes in BoDV-1-infected cells can be relieved with anti-BoDV-1 treatment. This study provides fundamental information regarding transcriptomic changes after BoDV-1 infection and the treatment in neuronal cells.

Borna disease virus 1 (BoDV-1) has been recognized as a rare cause of very severe encephalitis with rapid onset in central Europe. Data on cerebrospinal fluid (CSF) analysis have not yet been analyzed in detail. Here, we present the first study on CSF changes in BoDV-1 encephalitis. We retrospectively analyzed CSFs from 18 BoDV-1 encephalitis cases from Bavaria, Germany, an endemic region, from 1996 to 2021. Data were obtained through review of medical records and institutional databases. We found that white blood cell count (WBC) in CSF is elevated in 13 of our 18 patients at first examination (average 83.2 ± 142.3 leukocytes/μl) and cytology showed predominance of lymphocytes. Patients with typical symptoms of meningoencephalitis had higher WBC in first CSF analyzation (133.5 ± 163.1 vs 4.0 ± 3.2/μl; p = 0.065). BoDV-1 PCR of CSF is not always positive when tested (7 of 9 cases). Four of five patients tested showed a polyvalent reaction against multiple viruses in the CSF suggesting that BoDV-1 may trigger autoimmune mechanisms. CSF changes in BoDV-1 encephalitis seem similar to those of other viral encephalitis and at the beginning WBC can be normal in up to 28%, making the diagnosis even more challenging. All in all, BoDV-1 should be included in the diagnostic workup of patients with rapidly evolving and/or severe encephalitis and patients with severe neuropathy and secondary encephalopathy with and without CSF changes. Repeated CSF examinations as well as BoDV-1 serology and CSF PCR have to be considered in endemic areas., Competing Interests: Declaration of Competing Interest The authors declare no financial or other conflicts of interest related to the manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bornaviruses are RNA viruses with a mammalian, reptilian, and avian host range. The viruses infect neuronal cells and in rare cases cause a lethal encephalitis. The family Bornaviridae are part of the Mononegavirales order of viruses, which contain a nonsegmented viral genome. Mononegavirales encode a viral phosphoprotein (P) that binds both the viral polymerase (L) and the viral nucleoprotein (N). The P protein acts as a molecular chaperone and is required for the formation of a functional replication/transcription complex. In this study, the structure of the oligomerization domain of the phosphoprotein determined by X-ray crystallography is reported. The structural results are complemented with biophysical characterization using circular dichroism, differential scanning calorimetry and small-angle X-ray scattering. The data reveal the phosphoprotein to assemble into a stable tetramer, with the regions outside the oligomerization domain remaining highly flexible. A helix-breaking motif is observed between the α-helices at the midpoint of the oligomerization domain that appears to be conserved across the Bornaviridae. These data provide information on an important component of the bornavirus replication complex., (open access.)

More than 40 human infections with the zoonotic Borna disease virus 1 (BoDV-1) have been reported to German health authorities from endemic regions in southern and eastern Germany. Diagnosis of a confirmed case is based on the detection of BoDV-1 RNA or BoDV-1 antigen. In parallel, serological assays such as ELISA, immunoblots, and indirect immunofluorescence are in use to detect the seroconversion of Borna virus-reactive IgG in serum or cerebrospinal fluid (CSF). As immunopathogenesis in BoDV-1 encephalitis appears to be driven by T cells, we addressed the question of whether an IFN-γ-based ELISpot may further corroborate the diagnosis. For three of seven BoDV-1-infected patients, peripheral blood mononuclear cells (PBMC) with sufficient quantity and viability were retrieved. For all three patients, counts in the range from 12 to 20 spot forming units (SFU) per 250,000 cells were detected upon the stimulation of PBMC with a peptide pool covering the nucleocapsid protein of BoDV-1. Additionally, individual patients had elevated SFU upon stimulation with a peptide pool covering X or phosphoprotein. Healthy blood donors ( n = 30) and transplant recipients ( n = 27) were used as a control and validation cohort, respectively. In this pilot study, the BoDV-1 ELISpot detected cellular immune responses in human patients with BoDV-1 infection. Its role as a helpful diagnostic tool needs further investigation in patients with BoDV-1 encephalitis.

More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study ("BoDV-1 after solid-organ transplantation") to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.

In 2021, three encephalitis cases due to the Borna disease virus 1 (BoDV-1) were diagnosed in the north and east of Germany. The patients were from the states of Thuringia, Saxony-Anhalt, and Lower Saxony. All were residents of known endemic areas for animal Borna disease but without prior diagnosed human cases. Except for one recently detected case in the state of Brandenburg, all >30 notified cases had occurred in, or were linked to, the southern state of Bavaria. Of the three detected cases described here, two infections were acute, while one infection was diagnosed retrospectively from archived brain autopsy tissue samples. One of the acute cases survived, but is permanently disabled. The cases were diagnosed by various techniques (serology, molecular assays, and immunohistology) following a validated testing scheme and adhering to a proposed case definition. Two cases were classified as confirmed BoDV-1 encephalitis, while one case was a probable infection with positive serology and typical brain magnetic resonance imaging, but without molecular confirmation. Of the three cases, one full virus genome sequence could be recovered. Our report highlights the need for awareness of a BoDV-1 etiology in cryptic encephalitis cases in all areas with known animal Borna disease endemicity in Europe, including virus-endemic regions in Austria, Liechtenstein, and Switzerland. BoDV-1 should be actively tested for in acute encephalitis cases with residence or rural exposure history in known Borna disease-endemic areas.

Borna disease virus 1 (BoDV-1) strains attracted public interest by recently reported rare fatal encephalitis cases in Germany. Previously, human BoDV-1 infection was suggested to contribute to psychiatric diseases. Clinical outcomes (encephalitis vs. psychiatric disease) and epidemiology (zoonotic vs. human-to-human transmission) are still controversial. Here, phylogenetic analyses of 18 human and 4 laboratory strains revealed close genomic homologies both in distant geographical regions, and different clinical entities. Single unique amino acid mutations substantiated the authenticity of human strains. No matching was found with those of shrew strains in the same cluster 4, arguing against zoonosis. Opposite epidemiology concepts should be equally considered.

Determining the structural organisation of viral replication complexes and unravelling the impact of infection on cellular homeostasis represent important challenges in virology. This may prove particularly useful when confronted with viruses that pose a significant threat to human health, that appear unique within their family, or for which knowledge is scarce. Among Mononegavirales , bornaviruses (family Bornaviridae ) stand out due to their compact genomes and their nuclear localisation for replication. The recent recognition of the zoonotic potential of several orthobornaviruses has sparked a surge of interest in improving our knowledge on this viral family. In this work, we provide a complete analysis of the structural organisation of Borna disease virus 1 (BoDV-1) phosphoprotein (P), an important cofactor for polymerase activity. Using X-ray diffusion and diffraction experiments, we revealed that BoDV-1 P adopts a long coiled-coil α-helical structure split into two parts by an original β-strand twist motif, which is highly conserved across the members of whole Orthobornavirus genus and may regulate viral replication. In parallel, we used BioID to determine the proximal interactome of P in living cells. We confirmed previously known interactors and identified novel proteins linked to several biological processes such as DNA repair or mRNA metabolism. Altogether, our study provides important structure/function cues, which may improve our understanding of BoDV-1 pathogenesis.

Borna disease virus 1 (BoDV-1) is a neurotropic RNA virus belonging to the family Bornaviridae within the order Mononegavirales . Whereas BoDV-1 causes neurological and behavioral disorders, called Borna disease (BD), in a wide range of mammals, its virulence in humans has been debated for several decades. However, a series of case reports in recent years have established the nature of BoDV-1 as a zoonotic pathogen that causes fatal encephalitis in humans. Although many virological properties of BoDV-1 have been revealed to date, the mechanism by which it causes fatal encephalitis in humans remains unclear. In addition, there are no effective vaccines or antiviral drugs that can be used in clinical practice. A reverse genetics approach to generating replication-competent recombinant viruses from full-length cDNA clones is a powerful tool that can be used to not only understand viral properties but also to develop vaccines and antiviral drugs. The rescue of recombinant BoDV-1 (rBoDV-1) was first reported in 2005. However, due to the slow nature of the replication of this virus, the rescue of high-titer rBoDV-1 required several months, limiting the use of this system. This review summarizes the history of the reverse genetics and artificial replication systems for orthobornaviruses and explores the recent progress in efforts to rescue rBoDV-1.

Background: Borna disease virus 1 (BoDV-1) is a non-segmented, negative-strand RNA virus that persistently infects mammals including humans. BoDV-1 worldwide occurring strains display highly conserved genomes with overlapping genetic signatures between those of either human or animal origin. BoDV-1 infection may cause behavioral and cognitive disturbances in animals but has also been found in human major depression and obsessive-compulsive disorder (OCD). However, the impact of BoDV-1 on memory functions in OCD is unknown., Method: To evaluate the cognitive impact of BoDV-1 in OCD, event-related brain potentials (ERPs) were recorded in a continuous word recognition paradigm in OCD patients (n = 16) and in healthy controls (n = 12). According to the presence of BoDV-1-specific circulating immune complexes (CIC), they were divided into two groups, namely group H (high) and L (low), n = 8 each. Typically, ERPs to repeated items are characterized by more positive waveforms beginning approximately 250 ms post-stimulus. This "old/new effect" has been shown to be relevant for memory processing. The early old/new effect (ca. 300-500 ms) with a frontal distribution is proposed to be a neural correlate of familiarity-based recognition. The late old/new effect (post-500 ms) is supposed to reflect memory recollection processes., Results: OCD patients were reported to show a normal early old/new effect and a reduced late old/new effect compared to normal controls. In our study, OCD patients with a high virus load (group H) displayed exactly these effects, while patients with a low virus load (group L) did not differ from healthy controls., Conclusion: These results confirmed that OCD patients had impaired memory recollection processes compared to the normal controls which may to some extent be related to their BoDV-1 infection., (© 2022. The Author(s).)

Borna disease virus 1 (BoDV-1) is a non-segmented, negative-strand RNA virus that is characterized by persistent infection in the nucleus and low production of progeny virions. This feature impedes not only the harvesting of infectious viral particles from infected cells but also the rescue of high titres of recombinant BoDV-1 (rBoDV-1) by reverse genetics. Here, we demonstrate that exogenous expression of both matrix protein (M) and glycoprotein (G), which are constituents of the viral lipid envelope, significantly facilitates the formation of infectious particles and propagation of BoDV-1 without affecting its viral RNA synthesis. Furthermore, simultaneous transfection of M and G expression plasmids with N, P and L helper plasmids by reverse genetics drastically enhances the rescue efficiency of rBoDV-1. On the other hand, we also show that overexpression of M induces obvious cytotoxicity similar to that of other Mononegaviruses. Together with our recent report showing that excess expression of G induces aberrant accumulation of immature G, a potential stimulator of the host innate immune response, it is conceivable that BoDV-1 may suppress excess expression of M and G to reduce the cytopathic effect, thereby leading to maintenance of persistent infection. Our results contribute not only to the establishment of an efficient method to recover high-titre BoDV-1 but also to understanding the unique mechanism of persistent BoDV-1 infection.

Borna disease (BD), a frequently fatal neurologic disorder caused by Borna disease virus 1 (BoDV-1), has been observed for decades in horses, sheep, and other mammals in certain regions of Europe. The bicoloured white-toothed shrew (Crocidura leucodon) was identified as a persistently infected species involved in virus transmission. Recently, BoDV-1 attracted attention as a cause of fatal encephalitis in humans. Here, we report investigations on BoDV-1-infected llamas from a farm in a BD endemic area of Switzerland, and alpacas from holdings in a region of Germany where BD was last seen in the 1960s but not thereafter. All New World camelids showed apathy and abnormal behaviour, necessitating euthanasia. Histologically, severe non-suppurative meningoencephalitis with neuronal Joest-Degen inclusion bodies was observed. BoDV-1 was confirmed by immunohistology, RT-qPCR, and sequencing in selected animals. Analysis of the llama herd over 20 years showed that losses due to clinically suspected BD increased within the last decade. BoDV-1 whole-genome sequences from one Swiss llama and one German alpaca and-for comparison-from one Swiss horse and one German shrew were established. They represent the first published whole-genome sequences of BoDV-1 clusters 1B and 3, respectively. Our analysis suggests that New World camelids may have a role as a sentinel species for BoDV-1 infection, even when symptomatic cases are lacking in other animal species., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Borna disease virus (BoDV), a nonsegmented, negative-sense RNA virus, establishes persistent infection and replicates in the cell nucleus. Since BoDV genomic RNA exists as episomal RNA, the host genome is not invaded by BoDV infection. These unique features make BoDV a promising gene delivery system as an RNA virus-based episomal vector (REVec). Previously, the stable expression of genes of interest in vitro and in vivo using a REVec was reported. For the clinical application of a REVec, the fundamental properties under various physical and chemical conditions must be determined to develop purification processes, supply chains, and biosafety management. This study investigated the effects of the following conditions on the inducibility of transmission-defective ΔG-REVec: freeze-thaw cycles, dehydration, UV, temperature, pH, and reagents for virucides and laboratory experiments. Although the titer of ΔG-REVec was not influenced by the freeze-thaw process or 5 minute incubation at ≤50°C, ΔG-REVec was significantly inactivated by incubation at ≥70°C for 5 minutes. The induction titer of ΔG-REVec was decreased by long-term incubation, dehydration, and UV irradiation in a temperature- and time-dependent manner. ΔG-REVec was sensitive to lower pH and inactivated by chemical reagents under general conditions. These results provide important knowledge for developing the clinical use of REVec and biosafety management., (© 2021 The Societies and John Wiley & Sons Australia, Ltd.)

Persistent intranuclear infection is an uncommon infection strategy among RNA viruses. However, Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, maintains viral infection in the cell nucleus by forming structured aggregates of viral ribonucleoproteins (vRNPs), and by tethering these vRNPs onto the host chromosomes. To better understand the nuclear infection strategy of BoDV-1, we determined the host protein interactors of the BoDV-1 large (L) protein. By proximity-dependent biotinylation, we identified several nuclear host proteins interacting with BoDV-1 L, one of which is TRMT112, a partner of several methyltransferases (MTases). TRMT112 binds with BoDV-1 L at the RNA-dependent RNA polymerase domain, together with BUD23, an 18S ribosomal RNA MTase and 40S ribosomal maturation factor. We then discovered that BUD23-TRMT112 mediates the chromosomal tethering of BoDV-1 vRNPs, and that the MTase activity is necessary in the tethering process. These findings provide us a better understanding on how nuclear host proteins assist the chromosomal tethering of BoDV-1, as well as new prospects of host-viral interactions for intranuclear infection strategy of orthobornaviruses., (© 2021 The Societies and John Wiley & Sons Australia, Ltd.)

Borna disease virus (BoDV) is a neurotropic virus that causes several infections in humans and neurological diseases in a wide range of animals worldwide. BoDV-1 has been molecularly and serologically detected in many domestic and wild animals in Japan; however, the genetic diversity of this virus and the origin of its infection are not fully understood. In this study, we investigated BoDV-1 infection and genetic diversity in samples collected from animals in Hokkaido between 2006 and 2020. The analysis was performed by focusing on the P region of BoDV-1 for virus detection. The presence of BoDV-1 RNA was observed in samples of brain tissue and various organs derived from persistently infected cattle. Moreover, after inoculation, BoDV-positive brains were isolated from neonatal rats. The gene sequences of the P region of BoDV obtained from the rat brain were in the same cluster as the P region of the virus isolated from the original bovine. Thus, genetic variation in BoDV-1 was extremely low. The phylogenetic analysis revealed that BoDV-1 isolates obtained in this study were part of the same cluster, which suggested that BoDV-1 of the same cluster was widespread among animals in Hokkaido.

Background: The true burden and geographical distribution of human Borna disease virus 1 (BoDV-1) encephalitis is unknown. All detected cases so far have been recorded in Bavaria, southern Germany., Case Presentation: A retrospective laboratory and epidemiological investigation of a 2017 case of fatal encephalitis in a farmer in Brandenburg, northeast Germany, demonstrated BoDV-1 as causative agent by polymerase chain reaction, immunohistochemistry and in situ hybridization. Next-generation sequencing showed that the virus belonged to a cluster not known to be endemic in Brandenburg. The investigation was triggered by a recent outbreak of animal Borna disease in the region. Multiple possible exposures were identified. The next-of-kin were seronegative., Conclusions: The investigation highlights clinical awareness for human BoDV-1 encephalitis which should be extended to all areas endemic for animal Borna disease. All previously diagnosed human cases had occurred > 350 km further south. Further testing of shrews and livestock with Borna disease may show whether this BoDV-1 cluster is additionally endemic in the northwest of Brandenburg., (© 2021. The Author(s).)

Members of the family Bornaviridae produce enveloped virions containing a linear negative-sense non-segmented RNA genome of about 9 kb. Bornaviruses are found in mammals, birds, reptiles and fish. The most-studied viruses with public health and veterinary impact are Borna disease virus 1 and variegated squirrel bornavirus 1, both of which cause fatal encephalitis in humans. Several orthobornaviruses cause neurological and intestinal disorders in birds, mostly parrots. Endogenous bornavirus-like sequences occur in the genomes of various animals. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bornaviridae , which is available at ictv.global/report/bornaviridae.

Borna disease virus 1 (BoDV-1) is a nonsegmented, negative-strand RNA virus that infects mammals including humans. BoDV-1 strains occur globally, dominate the species Mammalian 1 bornavirus, and display highly conserved genomes and persistent infection (brain, blood). Subclinical infections prevail but the rare fatal outcomes even in people need awareness and risk assessment. Although BoDV-1 strains were successfully isolated, only limited full genomic sequences are available. In this study, the entire genomes of two natural BoDV-1 isolates (Hu-H2, Equ-Cres) and one vaccine strain (DessVac) were sequenced. They were compared with 20 genomes and 20 single-gene sequences (N and P) of worldwide human strains from psychiatric and neurologic patients and animal strains from horses with Borna disease available at GenBank. Phylogenetic analyses confirmed a low divergence not exceeding 5.55%, 5.34%, and 4.94% at the genome, P-gene, and N-gene level, respectively, characteristic of BoDV-1. Human viruses tended to cluster at the country level but appeared to be independent of hosts' diseases and/or time of isolation. Notably, our data also indicated that human viruses provided individual genetic signatures but exhibited no distinct genotypes that separated them from animal strains. Sequence similarities thus occurred between different host species and distant geographic regions, supporting global BoDV-1 prevalence. Overall low genetic divergence among BoDV-1 viruses shown here also argued against zoonotic concepts, requiring further clarification beyond sequence similarities. Finally, unlike shared sequence conservation, phenotyping of natural and laboratory variants revealed that they manipulated host cells differently, underpinning the authenticity of the human BoDV-1 strains., (© 2020 Wiley Periodicals LLC.)

Endogenous retroviruses have demonstrated exaptation during long-term evolution with hosts, e.g., resulting in acquisition of antiviral effect on related extant viral infections. While empirical studies have found that an endogenous bornavirus-like element derived from viral nucleoprotein (itEBLN) in the ground squirrel genome shows antiviral effect on virus replication and de novo infection, the antiviral mechanism, dynamics, and quantitative effect of itEBLN remain unknown. In this study, we experimentally and theoretically investigated the dynamics of how an extant bornavirus, Borna disease virus 1 (BoDV-1), spreads and replicates in uninfected, BoDV-1-infected, and itEBLN-expressing cultured cells. Quantifying antiviral effect based on time course data sets, we found that the antiviral effects of itEBLN are estimated to be 75% and 34% on intercellular virus spread and intracellular virus replication, respectively. This discrepancy between intercellular virus spread and intracellular viral replication suggests that viral processes other than the replication of viral ribonucleoprotein complex (RNP) contributed to the suppression of virus spread in itEBLN-expressing cells. Because itEBLN binds to the BoDV-1 RNP, the suppression of viral RNP trafficking can be an attractive candidate explaining this discrepancy. IMPORTANCE Accumulating evidence suggests that some endogenous viral elements (EVEs), including endogenous retroviruses and endogenous nonretroviral virus elements, have acquired functions in the host as a result of long-term coevolution. Recently, an endogenous bornavirus-like element (itEBLN) found in the ground squirrel genome has been shown to have antiviral activity against exogenous bornavirus infection. In this study, we first quantified bornavirus spread in cultured cells and then calculated the antiviral activity of itEBLN on bornavirus infection. The calculated antiviral activity of itEBLN suggests its suppression of multiple processes in the viral life cycle. To our knowledge, this is the first study quantifying the antiviral activity of EVEs and speculating on a model of how some EVEs have acquired antiviral activity during host-virus arms races., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In 2018-19, Borna disease virus 1 (BoDV-1), the causative agent of Borna disease in horses, sheep, and other domestic mammals, was reported in five human patients with severe to fatal encephalitis in Germany. However, information on case frequencies, clinical courses, and detailed epidemiological analyses are still lacking. We report the occurrence of BoDV-1-associated encephalitis in cases submitted to the Institute of Clinical Microbiology and Hygiene, Regensburg University Hospital, Regensburg, Germany, and provide a detailed description of newly identified cases of BoDV-1-induced encephalitis., Methods: All brain tissues from 56 encephalitis cases from Bavaria, Germany, of putative viral origin (1999-2019), which had been submitted for virological testing upon request of the attending clinician and stored for stepwise diagnostic procedure, were systematically screened for BoDV-1 RNA. Two additional BoDV-1-positive cases were contributed by other diagnostic centres. Positive results were confirmed by deep sequencing, antigen detection, and determination of BoDV-1-reactive antibodies in serum and cerebrospinal fluid. Clinical and epidemiological data from infected patients were collected and analysed., Findings: BoDV-1 RNA and bornavirus-reactive antibodies were detected in eight newly analysed encephalitis cases and the first human BoDV-1 isolate was obtained from an unequivocally confirmed human BoDV-1 infection from the endemic area. Six of the eight BoDV-1-positive patients had no record of immunosuppression before the onset of fatal disease, whereas two were immunocompromised after solid organ transplantation. Typical initial symptoms were headache, fever, and confusion, followed by various neurological signs, deep coma, and severe brainstem involvement. Seven of nine patients with fatal encephalitis of unclear cause were BoDV-1 positive within one diagnostic centre. BoDV-1 sequence information and epidemiological analyses indicated independent spillover transmissions most likely from the local wild animal reservoir., Interpretation: BoDV-1 infection has to be considered as a potentially lethal zoonosis in endemic regions with reported spillover infections in horses and sheep. BoDV-1 infection can result in fatal encephalitis in immunocompromised and apparently healthy people. Consequently, all severe encephalitis cases of unclear cause should be tested for bornaviruses especially in endemic regions., Funding: German Federal Ministry of Education and Research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The majority of emerging infectious diseases are zoonotic in nature and originate from wildlife reservoirs. Borna disease, caused by Borna disease virus 1 (BoDV-1), is an infectious disease affecting mammals, but recently it has also been shown to cause fatal encephalitis in humans. The endemic character of Borna disease points towards a nature-bound reservoir, with only one shrew species identified as reservoir host to date. Bats have been identified as reservoirs of a variety of zoonotic infectious agents. Endogenous borna-like elements in the genome of certain bat species additionally point towards co-evolution of bats with bornaviruses and therefore raise the question whether bats could serve as a potential reservoir of orthobornaviruses., Methods: Frozen brain samples (n = 257) of bats of seven different genera from Germany were investigated by orthobornaviral RT-PCR. Additionally, tissue slides of formalin-fixed paraffin-embedded material of a subset of these bats (n = 140) were investigated for orthobornaviral phosphoprotein by immunohistochemistry., Results: The brain samples were tested by RT-PCR without any evidence of orthobornavirus specific amplicons. Immunohistochemistry revealed a faint immunoreaction in 3/140 bats but with an untypical staining pattern for viral antigen., Conclusions: RT-PCR-screening showed no evidence for orthobornaviral RNA in the investigated bats. However, immunohistochemistry results should be investigated further to elucidate whether the reaction might be associated with expressed endogenous bornaviral elements or other so far unknown bornaviruses.

Knowledge on bornaviruses has expanded tremendously during the last decade through detection of novel bornaviruses and endogenous bornavirus-like elements in many eukaryote genomes, as well as by confirmation of insectivores as reservoir species for classical Borna disease virus 1 (BoDV-1). The most intriguing finding was the demonstration of the zoonotic potential of lethal human bornavirus infections caused by a novel bornavirus of different squirrel species (variegated squirrel 1 bornavirus, VSBV-1) and by BoDV-1 known as the causative agent for the classical Borna disease in horses and sheep. Whereas a T cell-mediated immunopathology has already been confirmed as key disease mechanism for infection with BoDV-1 by experimental studies in rodents, the underlying pathomechanisms remain less clear for human bornavirus infections, infection with other bornaviruses or infection of reservoir species. Thus, an overview of current knowledge on the pathogenesis of bornavirus infections focusing on immunopathology is given., (© 2020 Elsevier Inc. All rights reserved.)

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.

Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations. IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes., (Copyright © 2019 American Society for Microbiology.)

The 3'-untranslated region (UTR) of the non-segmented, negative-strand (NNS) RNA viral genome is called the leader sequence, and functions as the promoter for viral replication and transcription. NNS RNA viruses also use the sequence as a template to synthesize leader RNAs (leRNAs) with unknown functions. Borna disease virus (BDV) is unique because it establishes a persistent infection and replicates in the nucleus. No report has yet demonstrated the presence of leRNAs during BDV infection. Here, we report that BDV synthesizes leRNAs from the 3'-UTR of the genome. They started at position 5 in the 3'-UTR and ended by the transcription start signal of the nucleoprotein gene. The level of leRNA production is not correlated with the levels of viral replication and transcription. On the other hand, mutation of the 3'-UTR affects leRNA production. Our findings add a novel viral transcript to the BDV life cycle and shed light on BDV replication and/or transcription., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

Bornaviruses, non-segmented, negative-strand RNA viruses, are emerging agents with the potential for causing various types of neurological symptoms. Previous studies have shown that ribavirin, a nucleic acid analog with broad-spectrum antiviral activity, has a potent antiviral effect on infections with a mammalian bornavirus, Borna disease virus (BoDV-1), as well as avian bornaviruses. However, ribavirin-based treatment does not eliminate bornaviruses from persistently infected cells and viral replication resumes after treatment cessation. Therefore, the development of a novel effective anti-bornavirus treatment is needed. To identify such agents, we screened nucleoside/nucleotide mimetics for agents with anti-bornavirus activity. We used Vero cells infected with recombinant BoDV-1 carrying Gaussia luciferase to monitor BoDV-1 replication and found that favipiravir (T-705) is a potent inhibitor of BoDV-1 replication. T-705 suppressed BoDV-1 replication in a dose- and time-dependent manner during the observation period of 4 weeks. Notably, no increase in luciferase activity or in the number of BoDV-1-positive cells was detected in the at least 4 weeks following T-705 removal. Finally, we demonstrated that T-705 effectively suppressed viral replication of both BoDV-1 and an avian bornavirus, suggesting that T-705 may have a strong antiviral activity against a broad range of bornaviruses. Our findings provide a novel and effective option for treating persistent bornavirus infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Borna disease, a lethal infection with Borna disease virus-1 (BoDV-1), was diagnosed in four horses from Upper Austria in 2015 and 2016. All cases occurred in winter (two cases in February 2015 and two cases in December 2016), and the maximal distance of the affected stables was 17 km. To demonstrate whether the causative agent was also harbored by its reservoir host, the bicolored white-toothed shrew (Crocidura leucodon), 28 shrews from this geographic area were collected in 2015 and investigated for the presence of BoDV-1. The shrew species were identified according to taxonomic clues and molecular barcodes. Affected horses and all shrews were investigated using histology, immunohistochemistry (IHC) and reverse transcription PCR. The horses exhibited severe nonpurulent encephalitis. Large amounts of BoDV-1 antigen were identified in their CNS. Among the 28 shrews, nine were identified as C. leucodon and 13 as Sorex araneus (Common shrew; Eurasian shrew). Six C. leucodon (66.7%) and one S. araneus (7.7%) had BoDV-1 infections. In accordance with previous findings, the IHC of C. leucodon exhibited a high amount of viral antigen in many neural and extraneural tissues. By contrast, the single positive S. araneus had an exclusively neural staining pattern. Of all positive samples, whole-genome BoDV-1 sequences were generated. The acquired sequences of the affected shrews were not identical to each other and clustered around the sequences of the diseased horses belonging, surprisingly, to the German 'strain V' cluster.

Long-range axonal retrograde transport is a key mechanism for the cellular dissemination of neuroinvasive viruses, such as Borna disease virus (BDV), for which entry and egress sites are usually distant from the nucleus, where viral replication takes place. Although BDV is known to disseminate very efficiently in neurons, both in vivo and in primary cultures, the modalities of its axonal transport are still poorly characterized. In this work, we combined different methodological approaches, such as confocal microscopy and biochemical purification of endosomes, to study BDV retrograde transport. We demonstrate that BDV ribonucleoparticles (composed of the viral genomic RNA, nucleoprotein and phosphoprotein), as well as the matrix protein, are transported towards the nucleus into endocytic carriers. These specialized organelles, called signalling endosomes, are notably used for the retrograde transport of neurotrophins and activated growth factor receptors. Signalling endosomes have a neutral luminal pH and thereby offer protection against degradation during long-range transport. This particularity could allow the viral particles to be delivered intact to the cell body of neurons, avoiding their premature release in the cytoplasm.

RNA interference (RNAi) has emerged as a promising technique for gene therapy. However, the safe and long-term expression of small RNA molecules is a major concern for the application of RNAi therapies in vivo. Borna disease virus (BDV), a non-segmented, negative-strand RNA virus, establishes a persistent infection without obvious cytopathic effects. Unique among animal non-retroviral RNA viruses, BDV persistently establishes a long-lasting persistent infection in the nucleus. These features make BDV ideal for RNA virus vector persistently expressing small RNAs. Here, we demonstrated that the recombinant BDV (rBDV) containing the miR-155 precursor, rBDV-miR-155, persistently expressed miR-155 and efficiently silenced its target gene. The stem region of the miR-155 precursor in rBDV-miR-155 was replaceable by any miRNA sequences of interest and that such rBDVs efficiently silence the expression of target genes. Collectively, BDV vector would be a novel RNA virus vector enabling the long-term expression of miRNAs for RNAi therapies.

To favor their replication, viruses express proteins that target diverse mammalian cellular pathways. Due to the limited size of many viral genomes, such proteins are endowed with multiple functions, which require targeting to different subcellular compartments. One salient example is the X protein of Borna disease virus, which is expressed both at the mitochondria and in the nucleus. Moreover, we recently demonstrated that mitochondrial X protein is neuroprotective. In this study, we sought to examine the mechanisms whereby the X protein transits between subcellular compartments and to define its localization signals, to enhance its mitochondrial accumulation and thus, potentially, its neuroprotective activity. We transfected plasmids expressing fusion proteins bearing different domains of X fused to enhanced green fluorescent protein (eGFP) and compared their subcellular localization to that of eGFP. We observed that the 5-16 domain of X was responsible for both nuclear export and mitochondrial targeting and identified critical residues for mitochondrial localization. We next took advantage of these findings and constructed mutant X proteins that were targeted only to the mitochondria. Such mutants exhibited enhanced neuroprotective properties in compartmented cultures of neurons grown in microfluidic chambers, thereby confirming the parallel between mitochondrial accumulation of the X protein and its neuroprotective potential.-Ferré C. A., Davezac, N., Thouard, A., Peyrin, J. M., Belenguer, P., Miquel, M.-C., Gonzalez-Dunia, D., Szelechowski, M. Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential., (© FASEB.)

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that establishes persistent infection in the nucleus. Although BDV forms viral inclusion bodies, termed viral speckles of transcripts (vSPOTs), which are associated with chromatin in the nucleus, the host factors involved in the maintenance of vSPOTs remain largely unknown. In this study, we identified X-linked RNA-binding motif protein (RBMX) as a nuclear factor interacting with BDV nucleoprotein. Interestingly, knockdown of RBMX led to disruption of the formation of vSPOTs and reduced both transcription and replication of BDV. Our results indicate that RBMX is involved in the maintenance of the structure of the virus factory in the nucleus.

MicroRNAs (miRNAs) regulate gene expression by inhibiting transcription or translation and are involved in diverse biological processes, including development, cellular differentiation and tumor generation. miRNA microarray technology is a high‑throughput global analysis tool for miRNA expression profiling. Here, the hippocampi of four borna disease virus (BDV)‑infected and four non‑infected control neonatal rats were selected for miRNA microarray and bioinformatic analysis. Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) analysis was subsequently performed to validate the dysregulated miRNAs. Seven miRNAs (miR‑145*, miR‑146a*, miR‑192*, miR‑200b, miR‑223*, miR‑449a and miR‑505), showed increased expression, whereas two miRNAs (miR‑126 and miR‑374) showed decreased expression in the BDV‑infected group. By RT‑qPCR validation, five miRNAs (miR‑126, miR‑200b, miR‑374, miR‑449a and miR‑505) showed significantly decreased expression (P<0.05) in response to BDV infection. Biocarta pathway analysis predicted target genes associated with 'RNA', 'IGF1mTOR', 'EIF2', 'VEGF', 'EIF', 'NTHI', 'extrinsic', 'RB', 'IL1R' and 'IGF1' pathways. Gene Ontology analysis predicted target genes associated with 'peripheral nervous system development', 'regulation of small GTPase-mediated signal transduction', 'regulation of Ras protein signal transduction', 'aerobic respiration', 'membrane fusion', 'positive regulation of cell cycle', 'cellular respiration', 'heterocycle metabolic process', 'protein tetramerization' and 'regulation of Rho protein signal transduction' processes. Among the five dysregulated miRNAs identified by RT‑qPCR, miR‑126, miR‑200b and miR‑449a showed a strong association with nervous system development, cell differentiation, proliferation and apoptosis.