Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System.

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.

One Health in action: Investigation of the first detected local cluster of fatal borna disease virus 1 (BoDV-1) encephalitis, Germany 2022.

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).

Borna disease in an adult free-ranging Eurasian beaver (Castor fiber albicus).

Borna disease (BD) associated with a peracute bacterial septicaemia with Escherichia coli was diagnosed in an adult female, naturally infected, free-ranging Eurasian beaver of the subspecies Castor fiber albicus, clinically characterized by weight loss, depression, weakness and gurgled peristaltic sounds. The beaver was euthanized humanely. Necropsy and light microscopy revealed a non-purulent meningoencephalitis with typical mononuclear perivascular cuffs and parenchymal infiltrates. The diagnosis of BD was confirmed by detection of viral antigen and RNA by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was sequenced and cluster analysis revealed a close relationship between endemic clusters in Saxony-Anhalt. This is the first report of naturally occurring BD in a free-ranging Eurasian beaver.

HAND2 suppresses favipiravir efficacy in treatment of Borna disease virus infection.

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.

No evidence of an association of multiple sclerosis (MS) with Borna disease virus 1 (BoDV-1) infections in patients within an endemic region: a retrospective pilot study.

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.

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

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

Impact of Borna Disease Virus Infection on the Transcriptome of Differentiated Neuronal Cells and Its Modulation by Antiviral Treatment.

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.

First detected geographical cluster of BoDV-1 encephalitis from same small village in two children: therapeutic considerations and epidemiological implications.

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.

Human Infections with Borna Disease Virus 1 (BoDV-1) Primarily Lead to Severe Encephalitis: Further Evidence from the Seroepidemiological BoSOT Study in an Endemic Region in Southern Germany.

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.

IFN-γ-Based ELISpot as a New Tool to Detect Human Infections with Borna Disease Virus 1 (BoDV-1): A Pilot Study.

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.

Cerebrospinal fluid in Borna disease virus 1 (BoDV-1) encephalitis.

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.

Vaccination against Borna Disease: Overview, Vaccine Virus Characterization and Investigation of Live and Inactivated Vaccines.

(1) Background: Vaccination of horses and sheep against Borna disease (BD) was common in endemic areas of Germany in the 20th century but was abandoned in the early 1990s. The recent occurrence of fatal cases of human encephalitis due to Borna disease virus 1 (BoDV-1) has rekindled the interest in vaccination. (2) Methods: The full genomes of the BD live vaccine viruses "Dessau" and "Giessen" were sequenced and analyzed for the first time. All vaccination experiments followed a proof-of-concept approach. Dose-titration infection experiments were performed in rabbits, based on both cell culture- and brain-derived viruses at various doses. Inactivated vaccines against BD were produced from concentrated cell culture supernatants and investigated in rabbits and horses. The BoDV-1 live vaccine "Dessau" was administered to horses and antibody profiles were determined. (3) Results: The BD live vaccine viruses "Dessau" and "Giessen" belong to clusters 3 and 4 of BoDV-1. Whereas the "Giessen" virus does not differ substantially from field viruses, the "Dessau" virus shows striking differences in the M gene and the N-terminal part of the G gene. Rabbits infected with high doses of cell-cultured virus developed neutralizing antibodies and were protected from disease, whereas rabbits infected with low doses of cell-cultured virus, or with brain-derived virus did not. Inactivated vaccines were administered to rabbits and horses, following pre-defined vaccination schemes consisting of three vaccine doses of either adjuvanted or nonadjuvanted inactivated virus. Their immunogenicity and protective efficacy were compared to the BD live vaccine "Dessau". Seventy per cent of horses vaccinated with the BD live vaccine "Dessau" developed neutralizing antibodies after vaccination. (4) Conclusion: Despite a complex evasion of immunological responses by bornaviruses, some vaccination approaches can protect against clinical disease. For optimal effectiveness, vaccines should be administered at high doses, following vaccination schemes consisting of three vaccine doses as basic immunization. Further investigations are necessary in order to investigate and improve protection against infection and to avoid side effects.

Word recognition memory and serum levels of Borna disease virus specific circulating immune complexes in obsessive-compulsive disorder.

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.

Molecular epidemiology of human Borna disease virus 1 infection revisited.

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.

Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections.

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage.

miR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.

Borna disease virus 1 impairs DNA double-strand break repair through the ATR/Chk1 signalling pathway, resulting in learning and memory impairment in rats.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus that can establish persistent infection in the central nervous system and cause cognitive dysfunction in neonatally infected rats. However, the mechanisms that lead to this cognitive impairment remain unclear. DNA double-strand breaks (DSBs) and their repair are associated with brain development and cognition. If DNA repair in the brain is reduced or delayed and DNA damage accumulates, abnormal cognitive function may result. We generated a rat model of BoDV-1 infection during the neonatal period and assessed behavioural changes using the open field test and Morris water maze. The levels of DSBs were determined by immunofluorescence and comet assays. Western blotting assessed proteins associated with DNA repair pathways. The results showed that BoDV-1 downregulated the ATR/Chk1 signalling pathway in the brain, impairing DNA damage repair and increasing the number of DSBs, which ultimately leads to cognitive dysfunction. Our findings suggest a molecular mechanism by which BoDV-1 interferes with DNA damage repair to cause learning and memory impairment. This provides a theoretical basis for elucidating BoDV-1-induced neurodevelopmental impairment.

Human Borna disease virus 1 (BoDV-1) encephalitis cases in the north and east of Germany.

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.

New World camelids are sentinels for the presence of Borna disease virus.

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.

Borna disease virus phosphoprotein triggers the organization of viral inclusion bodies by liquid-liquid phase separation.

Inclusion bodies (IBs) are characteristic biomolecular condensates organized by the non-segmented negative-strand RNA viruses belonging to the order Mononegavirales. Although recent studies have revealed the characteristics of IBs formed by cytoplasmic mononegaviruses, that of Borna disease virus 1 (BoDV-1), a unique mononegavirus that forms IBs in the cell nucleus and establishes persistent infection remains elusive. Here, we characterize the IBs of BoDV-1 in terms of liquid-liquid phase separation (LLPS). The BoDV-1 phosphoprotein (P) alone induces LLPS and the nucleoprotein (N) is incorporated into the P droplets in vitro. In contrast, co-expression of N and P is required for the formation of IB-like structure in cells. Furthermore, while BoDV-1 P binds to RNA, an excess amount of RNA dissolves the liquid droplets formed by N and P in vitro. Notably, the intrinsically disordered N-terminal region of BoDV-1 P is essential to drive LLPS and to bind to RNA, suggesting that both abilities could compete with one another. These features are unique among mononegaviruses, and thus this study will contribute to a deeper understanding of LLPS-driven organization and RNA-mediated regulation of biomolecular condensates.