Multimodal Profiling of Immune Responses Reveals Innate-Adaptive Immune Imbalance in Human Bornavirus Encephalitis

Multimodal Profiling of Immune Responses Reveals Innate-Adaptive Immune Imbalance in Human Bornavirus Encephalitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multimodal Profiling of Immune Responses Reveals Innate-Adaptive Immune Imbalance in Human Bornavirus Encephalitis Nicola Jungbäck, Przemyslaw Grochowski, Daniel Hieber, Moritz Dinser, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9072167/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Human bornavirus encephalitis (BVE) is a rare, emerging and fatal zoonotic disease mainly caused by the Borna disease virus 1 (BoDV-1), a non-cytolytic RNA virus. Despite increasing recognition, the immunopathogenesis of human BoDV-1 infection remains unexplored. Complete coronary and sagittal brain sections from four fatal BoDV-1 cases were analysed using digitised immunohistochemistry to quantify viral distribution and tissue responses. Transcriptome-based analyses characterised local immune cell profiles in relation to viral loads measured by RT-qPCR. BoDV-1 viral loads varied substantially between cases but showed region-specific enrichment in the basal ganglia and hippocampus, correlating with lymphocyte presence and reactive microglia and astrocytes. Immune cell deconvolution revealed viral load-dependent modulation dominated by innate immune and glial populations, including metabolic and reactive astrocyte states, IFNγ-responsive microglia, and dendritic cells, macrophages, neutrophils, basophils, and CD8⁺ T cells. This was accompanied by induction of interferon-stimulated genes, antigen presentation, protein synthesis, and oxidative stress pathways, with a transcriptional signature resembling non-lytic viral and autoimmune-like neuroinflammatory conditions rather than lytic infections. These findings support a model of BoDV-1 encephalitis characterised by a dominant innate immune response and delayed adaptive immune engagement, which may contribute to ineffective viral clearance and extensive tissue damage. BoDV-1 encephalitis neurotropic virus immunopathogenesis transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Human bornavirus encephalitis (BVE) is a rare but mostly fatal disease. While the main causative pathogen, the Borna disease virus 1 (BoDV-1), has been known to infect animals for decades, the first confirmed human cases were only identified in 2018 [17]. Since then, BoDV-1 has been recognised as a significant zoonotic pathogen in the endemic areas in Southern and Eastern Germany [3]. Approximately five to ten new human cases are confirmed each year, with a cumulative total of around 50 known cases to date [13].Clinically, BoDV-1 infection often begins with nonspecific, flu-like symptoms, followed by rapid neurological deterioration with altered consciousness, seizures, and motor deficits. In most cases, the disease progresses swiftly, leading to death within a few weeks after symptom onset [6, 13, 23]. The combination of high case fatality, rapid progression, and diagnostic challenges underscore the urgent need for increased clinical awareness and further therapeutic research, while also positioning BVE as valuable model for studying the pathogenesis of highly neurotropic viruses in general. Our initial neuropathological investigations described the first histological characteristics of BVE in six human autopsy cases. The viral distribution pattern showed prominent involvement of the basal ganglia with histological definition as a sclerosing panencephalitis with lymphocytic infiltration, microglial nodules, and intranuclear eosinophilic inclusions [7]. Subsequent findings revealed the presence of BoDV-1 in endothelial cells, observed under certain conditions and linked to vascular injury caused by hypoxic stress [8]. Finck et al. extended the understanding of BVE by correlating magnetic resonance imaging (MRI) findings with histopathological changes in a separate set of cases. They identified a reproducible pattern of radiological changes, beginning in the caudate nucleus and insular cortex, followed by involvement of the limbic system and brainstem [4]. Although the exact immunopathological mechanisms remain unclear to date, evidence suggests that BoDV-1 is non-cytolytic, with tissue injury primarily immune-mediated, involving CD4⁺ and CD8⁺ T cells, as well as reactive astrocytes, which are increasingly recognised as active contributors to disease progression [11, 14]. In selected cases, immunosuppressive treatment has been associated with prolonged survival, underscoring the potential role of immune modulation in disease management [6, 14]. Serum and cerebrospinal fluid analysis from ten individuals with BVE demonstrated a predominantly proinflammatory cytokine profile, indicating sustained recruitment of immune cells. Such an inflammatory milieu may also disrupt astrocyte function, potentially resulting in neuronal excitotoxicity [14]. Although individual immunopathological features of BVE have been described, the spatial viral distribution within the human brain and the neuroimmune responses remain insufficiently characterised. To address these gaps, we systematically analysed BoDV-1 dissemination in fully embedded, anatomically preserved brain cross-sections, and performed in-depth, transcriptome-based profiling of host neuroimmune responses. We hypothesised that BoDV-1 infection induces a distinct neuroinflammatory response shaped by viral tropism and local tissue context. Beyond advancing the understanding of BVE pathogenesis, our findings support the use of BVE as model to study host-pathogen interactions in infections caused by neurotropic viruses. Materials and Methods Material Brain autopsy material from four individuals (ages 39-71 years; median 68.5 years; all female) who died of BVE between 2022 and 2024 was included. Cases 3 and 4 were previously described [2, 23], whereas Cases 1 and 2 have not been published yet. For Cases 1 and 2, complete sagittal sections of the right hemisphere and complete coronal sections of the left hemisphere were prepared. Case 3 included a right sagittal section; Case 4 a coronal section through both hemispheres to assess viral distribution symmetry. Cross-sections were subdivided into approximately equally sized blocks and embedded into capsules, allowing for later reconstruction of the whole-brain slices. In total, 157 formalin-fixed, paraffin-embedded (FFPE) blocks were included in the analyses (median 37 blocks per case; range 31 to 52). Control tissue from a 64-year-old woman who died of toxic cardiocirculatory failure in the setting of advanced metastatic breast carcinoma and pleural effusion included two FFPE blocks (left thalamus and frontal cortex). Ethical approval was obtained from the ethics committee of the Ludwig-Maximilians-University ethics committee, responsible for Augsburg University Hospital (approval no. 23-0267). General Study Design and Workflow As outlined in Figure 1, all FFPE blocks were stained with hematoxylin and eosin (HE) and BoDV-1 immunohistochemistry (IHC) using automated staining systems (HE: Tissue-Tek Prisma, Sakura, Japan; IHC: Leica Bond RX, Leica Biosystems, Germany). In Case 4, additional immunostainings for T cell marker CD3, B cell marker CD20, microglia marker Iba-1, and astrocytic marker GFAP were performed. A total of 450 slides were prepared and digitised (scanner Pannoramic II, 3DHISTECH, Hungary). Sagittal and coronary cross-sections were digitally reconstructed. Based on BoDV-1 immunoreactivity, automated signal quantification analysis using the QuPath software [1] was performed, generating a histopathological (H-)Score for each FFPE block. Additionally, CellQuant software (3DHISTECH, Hungary) was used for enhanced visualisation of BoDV-1 signal distribution. RNA was separately extracted from all blocks to determine BoDV-1 viral loads. For transcriptomic profiling, 12 FFPE blocks per case (covering low, medium, and high viral loads; total: 50 samples, incl. two controls) were analysed using the nCounter Sprint Profiler (NanoString/Bruker, United States) to characterise immunological gene expression profiles. The resulting data were subjected to statistical analysis using custom Python (Python Software Foundation, United States) workflows. Details on all methods are provided in the Supplementary Material. Results Regional BoDV-1 Viral Load Profiling Reveals Marked Interindividual Variability but Consistent Hotspots in Deep Brain Structures Coronal and sagittal brain sections were analysed using three complementary approaches (Fig. 2A): BoDV-1 immunoreactivity was assessed via CellQuant-based digital visualisation and semi-quantitative H-Scoring using QuPath (see Supplementary Material Table 1 ). In parallel, BoDV-1 RNA levels were determined by real-time quantitative polymerase chain reaction (RT-qPCR) across all FFPE blocks. For the standardised anatomical assignment of tissue blocks see Supplementary Material Figure 1 . Viral loads were subsequently classified into low, medium, and high categories, first within each individual case and then across all cases. Due to marked interindividual variability, distinct thresholds were applied in each context. Mean viral loads differed interindividually by several orders of magnitude with ranges from 170.37 copies(c)/ng RNA in Case 4 to 462 711.72 c/ng RNA in Case 3. Case 1 (94 998.83 c/ng RNA) and Case 2 (9 343.39 c/ng RNA) showed intermediate values. Across all cases, FFPE blocks containing the basal ganglia, hippocampus, thalamus, and brainstem consistently showed higher viral loads. This regional pattern was mirrored by immunohistochemistry: H-Score analysis revealed the highest mean score in Case 3 (54.35), and the lowest in Case 4 (6.11), with intermediate values in Cases 1 (36.23) and 2 (46.77; Supplementary Material Tables 2 and 3 ). BoDV-1 RNA levels and antigen detection showed weak but statistically significant positive correlations (Pearson’s ρ = 0.317, p < 0.001; Spearman’s ρ = 0.244, p = 0.002), consistent with a parallel distribution of viral RNA and immunoreactivity for BoDV-1 nucleoprotein. Bilateral Viral Load Comparison Shows Symmetric Distribution in Case 4 In Case 4, paired measurements of viral load revealed a highly symmetric distribution between both hemispheres. Pearson correlation analysis demonstrated a strong positive linear correlation (ρ = 0.951, p < 0.001) between the respective FFPE blocks, supporting a symmetric viral spread. This was corroborated by a paired t-test, which showed no significant difference between hemispheres (t = -0.974, p = 0.3480). Correlation Analysis Identifies Coordinated T Cell, Microglial, and Astrocytic Responses to BoDV-1 RNA Levels For Case 4, an intra-case correlation analysis was performed to explore associations between BoDV-1 levels and cellular immune markers. Spearman correlation coefficients were calculated for BoDV-1 RNA levels, and the expression levels of CD3 (T cells), CD20 (B cells), Iba-1 (microglia), and GFAP (astrocytes). The analysis revealed several significant correlations ( Supplementary Material Figure 2 ). BoDV-1 RNA levels showed a moderate positive correlation with CD3-positive cells (ρ = 0.587, p < 0.001) and correlated moderately with Iba-1 (ρ = 0.502, p = 0.003), and GFAP (ρ = 0.406, p = 0.019), according to the classification by Schober et al. [19] . Additionally, all pairwise combinations of CD3, Iba-1, and GFAP showed significant positive correlations (CD3/GFAP: ρ = 0.493, p = 0.003; CD3/Iba-1: ρ = 0.403, p = 0.018; Iba-1/GFAP: ρ= 0.480, p = 0.004). Transcriptomic Immune Cell Mapping Suggests Viral Load-dependent Modulation of Immune and Glial Populations To systematically assess the relationship between viral burden and immune activation, all tissue samples were stratified into three groups (BoDV-1 high , BoDV-1 medium , BoDV-1 low ) based on viral RNA copy numbers (thresholds listed in Fig. 2A). A non-infected control served as reference. In total, 50 tissue samples were selected for transcriptome-based profiling using the nCounter platform, providing high-resolution insights into the immune microenvironment across varying viral loads in different brain areas. Immune cell composition was inferred by transcriptomic deconvolution using cell-type-specific gene signatures and pathways, covering not only brain-specific cell types such as astrocytes and microglia, but also immune cells attracted to the site of inflammation. The aim was to determine whether distinct immune cell subsets are differentially enriched across varying levels of viral loads. The marked interindividual differences in viral burden necessitated an additional intra-case evaluation for Case 3, which exhibited by far the highest viral RNA levels. As shown in Figure 2A, the thresholds for viral load categories were accordingly adapted. Across all BoDV-1-infected brain samples, a clear viral load-dependent modulation of immune cell populations was observed ( Supplementary Material Figure 3A ). Dendritic cells emerged as the most abundant population under all viral load groups. Macrophages were the second most prominent population, which seems consistent with their role in phagocytic clearance and cytokine release. Basophils and neutrophils showed moderate enrichment, which likely reflects a secondary influx in response to tissue damage and inflammation. Other immune subsets, including monocytes, natural killer (NK) cells, and T cells, exhibited a similar abundance in response. Plasma cells, eosinophils, B cells, mast cells, and myeloid-derived suppressor cells, were only sparsely detected, indicating a limited role in the dominant inflammatory response. Although T cells were not the most dominant immune population, they play a crucial role in the immune response to BoDV-1 infection, and, exhibiting cuffing around blood vessels and tissue infiltration are a histopathological hallmark of encephalitis. A more detailed analysis of T cell subsets revealed that CD8⁺ T cells were the most abundant across all BoDV-1 viral load groups, followed by CD4⁺ and memory T cells ( Supplementary Material Figure 3B ). The distribution followed a similar pattern, with the low groups exhibiting slightly higher values than the medium viral load groups, particularly for the CD8⁺ T cells. For CD4⁺ and memory T cells, the distribution was relatively even across all groups. A closer look at the astrocyte and microglia subsets revealed a similar distribution pattern with predomination of the disease-associated subtype ( Supplementary Material Figure 3 C/D ). In astrocytes, this was followed by the metabolic and reactive subtypes, while in microglia, the interferon-γ-induced subtype ranked second. These findings suggest a strong association between the viral load and the activation of specific immune and glial cell subsets. To minimise variability between individuals and confirm the findings under more controlled conditions, a within-case analysis was repeated for Case 3. Notably, the results closely resembled the patterns seen across all BoDV-1 viral load groups, supporting the homogeneity of the observed immune profile. Dendritic cells again emerged as the predominant population, irrespective of local viral load (Fig. 2B/C), followed by macrophages. Neutrophils and basophils were also repeatedly detected, indicating innate immune recruitment in response to infection or tissue damage. Although monocytes, NK cells, and T cells appeared less frequently, they were consistently present ( Supplementary Material Figure 4A ). A more detailed analysis of T cells, astrocytes, and microglia confirmed this trend: CD8⁺ T cells were again by far the most dominant subset and their relative abundance slightly decreased in BoDV-1 medium regions ( Supplementary Material Figure 4B ), accompanied by a marked accumulation of disease-associated astrocytes ( Supplementary Material Figure 4C ) and microglia ( Supplementary Material Figure 4D ). Pathway and Pseudotime Analysis Reveal Load-dependent Innate Activation and Limited Adaptive Immune Engagement Across all BoDV-1-infected brain samples, transcriptomic patterns across different viral load levels were dominated by innate immune signalling pathways, with progressive upregulation of interferon-stimulated genes (ISGs), antigen presentation, protein synthesis, and stress-response pathways ( Supplementary Material Figure 5A ) indicating dose-dependent activation of type I interferon signalling via pattern recognition receptors, a defining feature of acute antiviral immunity [18]. The within-case analysis of Case 3 ( Supplementary Material Figure 5B ) mirrored these patterns, showing the same ISG- and antigen-presentation-driven response with similar distribution across viral load groups, despite minor shifts in the relative ranking of pathways. The pathway-level expression network revealed a structured immune response aligned with viral load (Fig. 3A). In BoDV-1 encephalitis, pathway clustering across pseudotime reflected a progression from early cellular maintenance to advanced immune activation. Low viral load tissue was dominated by Notch signalling, which occupied an isolated position at the early end of the pseudotime axis. Medium viral load samples were associated with DNA repair, protein synthesis, signal transduction, angiogenesis, and Wnt signalling, forming a central cluster indicative of early-to-intermediate cellular activation. In contrast, high viral load samples displayed dense clustering of stress- and immune-related pathways. Of note, adaptive immune pathways were located at the interface between medium and high viral load clusters, suggesting a transitional or delayed engagement of effector responses. Control samples did not form a dominant cluster, underscoring their limited role in the overall immune activation profile. In Case 3, low and high viral load samples dominated the expression landscape (Fig. 3B). Low viral load was associated with early response pathways such as DNA repair, adaptive immunity, and angiogenesis, whereas high viral load corresponded to robust activation of innate immunity, oxidative stress, inflammation, and immune checkpoints. Notably, adaptive immune signalling formed no coherent cluster, suggesting a limited transition to effective T cell-mediated responses despite persistent viral presence. Gene Expression Analysis Shows Threshold-dependent Activation of Innate and Inflammatory Pathways Transcriptomic profiling across all four cases revealed viral load-dependent immune signatures. Differential gene expression was most pronounced in comparisons involving BoDV-1 high samples (vs. BoDV-1 medium and vs. BoDV-1 low ; Fig. 4), supporting that substantial immune activation occurs only above a critical viral threshold. In BoDV-1 high tissue, the marked upregulation of CD68, IL1B, and CCL3 indicated pronounced microglial activation and proinflammatory signalling. The consistent increase in CASP8 expression further suggested an engagement of inflammatory cell death pathways under high viral load conditions. Compared to uninfected controls, BoDV-1 high samples further showed increased expression of CCR5, supporting an enhanced chemokine-mediated immune cell recruitment to the CNS [21]. Interestingly, CXCL9, a pro-inflammatory chemokine involved in T cell recruitment, was significantly underexpressed in BoDV-1 medium compared to BoDV-1 low samples (Fig. 4B). In Case 3, BoDV-1 high samples exhibited pronounced transcriptional changes consistent with strong antiviral immune activation ( Supplementary Material Figure 6 ). Most significantly upregulated genes included CD68 (activated microglia/macrophages), TLR2 (sensor of viral ligands), OAS1 (interferon-stimulated antiviral effector), and CXCL10 (T cell recruiting chemokine), reflecting coordinated activation of innate immune sensors and CNS-resident effector cells. Upregulation of FCGR3 supports a role for antibody-dependent innate mechanisms [10]. Comparison between medium and low viral load revealed only minor changes. Compared to controls, BoDV-1 high samples showed increased CASP3 and CCR5, indicating enhanced cytotoxicity and leukocyte recruitment [21]. Pathway-based Gene Set Analysis Reveals Upregulation of Non-lytic Viral and Autoimmune-like Signatures A Kyoto Encyclopedia of Genes and Genomes (KEGG)-based gene set enrichment analysis (GSEA), including neurotropic, inflammatory, and non-pathogen-associated encephalitic reference sets, revealed a structured and category-specific transcriptional response across BoDV-1 viral load groups (Fig. 5). Compared to control tissue, the signatures for herpes simplex virus 1 (HSV-1), measles and Epstein-Barr virus were downregulated across all groups. In contrast, the signatures for the neurotropic tick-borne virus, rabies virus (RV), varicella-zoster virus and JC-virus (causative agent of progressive multifocal leukoencephalopathy) were uniformly upregulated. Interestingly, signatures for autoimmune diseases, such as multiple sclerosis and acute disseminated encephalomyelitis also showedupregulation. Discussion BVE is an emerging, severe and mostly fatal inflammatory disease of the central nervous system without any established treatment regimen. Neuropathological studies describe a lymphocyte-rich panencephalitis with widespread lesions showing intranuclear inclusions in neurons and glial cells, microglial nodule formation, astrocyte activation, and severe tissue damage primarily driven by a strong proinflammatory immune response. Although involvement of CD4 + /CD8 + T cells, microglial and astrocytic activation, and proinflammatory cytokine production has been described [7, 11, 14], the associated neuroimmune responses in the human brain remain poorly understood. This study aimed to address this gap through anatomically resolved viral mapping and transcriptome-based immune profiling. Our results show that BoDV-1 viral loads varied markedly between individuals, likely reflecting differences in treatment regimens rather than purely biological factors. High viral burdens in the basal ganglia, hippocampus, thalamus, and brainstem are consistent with previous reports [4, 5, 7]. Correlation between BoDV-1 RNA levels and immune cell and glial markers suggest an association of viral replication with T cell infiltration and reactive activation of microglia and astrocytes, features prominently observed in neuropathological examinations of BVE cases [7, 14]. Immune cell populations in BoDV-1-infected brain samples displayed distinct activation patterns linked to viral load levels. Local dendritic cells together with attracted macrophages, neutrophils, and basophils were the most abundant immune subsets, while CD8⁺ T lymphocytes dominated the adaptive compartment, consistent with Rauch et al. [14]. Although a moderate positive correlation between viral RNA levels and CD3⁺ T cell abundance was observed, stratification by viral load indicates a more nuanced, potentially stage-specific regulation of cytotoxic T cell responses. The activation of ISGs, antigen presentation, protein synthesis, and oxidative stress pathways reflects a robust antiviral and cellular stress response. Notably, our data suggest a relative underrepresentation of adaptive immune signalling, which may reflect either a temporal delay in adaptive responses or active suppression mechanisms. This aligns with established processes of immune regulation, including T cell exhaustion, checkpoint inhibition, and pathogen-driven immune evasion [24]. Altogether, the findings indicate a tiered immune response in BVE, shifting from homeostatic and repair-associated states towards antiviral and stress-driven activation as viral loads increase. This constellation, characterised by a strong innate response alongside lower or suppressed adaptive immunity, may help explain why BoDV-1 is not cleared despite pronounced inflammation and severe tissue destruction, thereby likely contributing to the fatal disease course. Importantly, these data do not contradict the established concept of immune-mediated pathology in BVE, but highlight the complexity of immune regulation in advanced stages. Immune activation occurred only above a critical viral threshold. Higher viral loads correlated with microglial activation, proinflammatory signalling, and cell death. Upregulation of CD68, IL1B, CCL3, CASP8, and CCR5, alongside downregulation of CXCL9 suggests a BoDV-1-driven immune environment that may limit antiviral T cell recruitment while stimulating microglia activation, potentially contributing to viral persistence. This finding highlights the need for a more detailed characterisation of microglia and their role during BoDV-1 infection. When comparing with other neurotropic virus infections, such as HSV-1 and RV infection, overlapping immune features were observed in BVE. Both, BoDV-1 and HSV-1 infection show CD68 and CXCL10 upregulation [20, 22]. IL1B is also upregulated in RV-infected microglia [9]. However, notable differences emerged from our analyses. HSV-1 infection primarily leads to CCL2 and CCL5 upregulation [20], whereas BoDV-1 infection selectively induced CCL3. No human data on CXCL9 expression exist for HSV-1 or RV, with insights into its functional role during HSV-1 infection derived solely from animal models [25]. Additionally, BVE showed transcriptional activation of apoptosis-related genes (CASP8, CASP9, LC3A), while during HSV-1 infection, these are mainly regulated at the post-translational level; for RV, such data are lacking. These differences highlight virus-specific immunopathological mechanisms despite shared innate immune activation, suggesting that each virus engages distinct strategies to shape host responses, and influence disease outcome. This is corroborated by KEGG enrichment analyses, which showed little overlap between BoDV-1 infection and transcriptional programs characteristic of cell-lytic viruses like HSV-1. This may explain the differences despite shared activation of innate immune pathways including oxidative stress responses, autophagy regulation, and antigen presentation [12, 15, 16]. In contrast, gene signatures associated with non-lytic neurotropic viruses, such as RV and tick-borne encephalitis virus, were upregulated in BoDV-1 infection. Interestingly, gene sets related to autoimmune neuroinflammatory conditions were likewise enriched. This combination may indicate a uniquely dysregulated neuroimmune state, aligning with previous clinical and neuropathological observations, including corticosteroid-responsive inflammation and prominent lymphocytic involvement, as reported previously [5, 14, 23]. Despite the novelty of our investigations of the immunopathogenesis of BVE, some challenges and limitations have to be acknowledged. Human BVE is an extremely rare disease, thereby limiting the number of cases available for analysis. Moreover, heterogenous disease courses and treatment attempts, together with the terminal-stage autopsy material, differences in post-mortem interval, and tissue fixation time, limit generalisability. However, even with these constraints, our data offer valuable insights into the immunopathogenesis of BVE. Collectively, the data indicate an imbalance characterised by robust activation of immune pathways in the presence of comparatively weak adaptive immune responses. Excessive innate immune activation may contribute to tissue injury and thereby limit effective immune control, potentially facilitating viral persistence. In line with this, pathway analyses showed little overlap with transcriptional programs of lytic viral infections but strong similarity to signatures of non-lytic neurotropic viruses and autoimmune neuroinflammation. Notably, animal models have demonstrated that impaired or immature immune responses can prevent disease manifestation, highlighting the critical role of host immune regulation in BoDV-1 encephalitis. Together, these findings suggest that adaptive immune mechanisms may contribute more substantially to BoDV-1 disease pathogenesis rather than the response being exclusively innate, and therefore warrant consideration in future therapeutic strategies. Declarations Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was granted by the ethics committee of the Ludwig-Maximilians-University, responsible for Augsburg University Hospital (approval no. 23-0267). Consent to participate The study used pseudonymised data and materials. Therefore, informed consent to participate was not required. Informed consent for autopsy was obtained from legal guardians. Consent to publish All data are fully anonymised, and no identifying information is included. Therefore, consent for publication was not required. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author. The authors confirm that data supporting the findings of this study are also available in the Supplementary Material. Funding FLS receives funding from the Deutsche Forschungsgemeinschaft (504757758), support from the Bavarian State Ministry of Health, Care, and Prevention (Zoonotic Bornavirus Focalpoint Bavaria [ZooBoFo]), and has received honorarium for a scientific presentation on bornavirus encephalitis at Ingolstadt Hospital (Ingolstadt, Germany). FLS and PA were supported by the Kurscheid-Hühnlein-Stiftung. None of the funding bodies were involved in either the design of the study, nor the collection, analysis, and interpretation of data. Acknowledgements The authors would like to express their sincere gratitude to Mrs. Natalia Gerling and Mrs. Juliane Stephan for their excellent conduct of the laboratory work. Figure 1 and Figure 2B were created with BioRender.com. The BoDV-1 antibody Bo18 was kindly provided by the Friedrich-Loeffler-Institute. 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Anesth Analg 126: 1763-1768 Doi 10.1213/ane.0000000000002864 Sehl-Ewert J, Schwaiger T, Schäfer A, Hölper JE, Klupp BG, Teifke JP, Blohm U, Mettenleiter TC (2022) Clinical, neuropathological, and immunological short- and long-term feature of a mouse model mimicking human herpes virus encephalitis. Brain pathology (Zurich, Switzerland) 32: e13031 Doi https://doi.org/10.1111/bpa.13031 Sorce S, Myburgh R, Krause KH (2011) The chemokine receptor CCR5 in the central nervous system. Prog Neurobiol 93: 297-311 Doi 10.1016/j.pneurobio.2010.12.003 Uyar O, Laflamme N, Piret J, Venable M-C, Carbonneau J, Zarrouk K, Rivest S, Boivin G (2020) An Early Microglial Response Is Needed To Efficiently Control Herpes Simplex Virus Encephalitis. Journal of Virology 94: 10.1128/jvi.01428-01420 Doi doi:10.1128/jvi.01428-20 Vollmuth Y, Jungbäck N, Mögele T, Schmidt-Graf F, Wunderlich S, Schimmel M, Rothe C, Stark L, Schlegel J, Rieder Get al (2024) Comparative Study of Virus and Lymphocyte Distribution with clinical Data suggests early high dose Immunosuppression as potential Key Factor for the Therapy of Patients with BoDV-1 Infection. Emerging microbes & infections: Doi https://doi.org/10.1080/22221751.2024.2350168 Wherry EJ, Kurachi M (2015) Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15: 486-499 Doi 10.1038/nri3862 Wuest T, Farber J, Luster A, Carr DJ (2006) CD4+ T cell migration into the cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice following herpes simplex virus type 1 infection. Cell Immunol 243: 83-89 Doi 10.1016/j.cellimm.2007.01.001 Additional Declarations Competing interest reported. FLS receives funding from the Deutsche Forschungsgemeinschaft (504757758), support from the Bavarian State Ministry of Health, Care, and Prevention (Zoonotic Bornavirus Focalpoint Bavaria [ZooBoFo]), and has received honorarium for a scientific presentation on bornavirus encephalitis at Ingolstadt Hospital (Ingolstadt, Germany). FLS and PA were supported by the Kurscheid-Hühnlein-Stiftung. Supplementary Files SUBMITJungbackAdditionalfileActaNeuropathologicaCommunications.docx SUBMITJungbackFiguresActaNeuropathologicaCommunications.pptx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Apr, 2026 Reviews received at journal 03 Apr, 2026 Reviews received at journal 28 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers agreed at journal 14 Mar, 2026 Reviewers invited by journal 13 Mar, 2026 Editor assigned by journal 12 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 09 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9072167","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606473009,"identity":"f584df43-a18d-478f-8931-5441832a8d40","order_by":0,"name":"Nicola Jungbäck","email":"","orcid":"","institution":"University Hospital Ulm","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Jungbäck","suffix":""},{"id":606473010,"identity":"0c0a45c7-c66c-4fe9-acfb-f63bf0a58a32","order_by":1,"name":"Przemyslaw Grochowski","email":"","orcid":"","institution":"University of 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dennis","middleName":"","lastName":"Tappe","suffix":""},{"id":606473028,"identity":"364f1be3-521b-4b17-aeab-8b76f1ebac8e","order_by":14,"name":"Friederike Liesche-Starnecker","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital Ulm","correspondingAuthor":true,"prefix":"","firstName":"Friederike","middleName":"","lastName":"Liesche-Starnecker","suffix":""}],"badges":[],"createdAt":"2026-03-09 11:23:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9072167/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9072167/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876625,"identity":"f8e75374-a672-4fa4-b8c7-39779e6a8650","added_by":"auto","created_at":"2026-03-18 08:43:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1440854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOutline of the general project framework\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/9c1c2497bc2caa7158da79bc.png"},{"id":104876728,"identity":"f7a1e67c-4477-4db9-af39-f4ab0813bf89","added_by":"auto","created_at":"2026-03-18 08:43:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10493737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIntegrated spatial analysis of viral distribution and immune cell landscape in BoDV-1-infected human brain tissue.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;Whole brain sections were evaluated using CellQuant-based BoDV-1 distribution visualisation, semi-quantitative H-Score assessment of BoDV-1 immunohistochemistry, and viral load quantification. Colour coding for H-Score and viral loads reflects the intensity of BoDV-1 detection: dark blue indicates high levels, royal blue medium levels, and light blue low levels. Due to marked interindividual variability, viral load classification was performed within each individual case (3\u003c/em\u003e\u003csup\u003e\u003cem\u003erd\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e column) and across all cases (4\u003c/em\u003e\u003csup\u003e\u003cem\u003eth\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e column). Blocks marked in red indicate those selected for downstream transcriptomic profiling. Threshold values for viral loads in copies/ng RNA.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Sagittal brain section of Case 3 illustrating regional viral load distribution. Areas with high viral load are shown in dark blue, medium viral load in royal blue, and low viral load in light blue. The size of each depicted immune cell type reflects its relative abundance in the respective region, as determined by transcriptomic analysis. Only the five most abundant immune cell population per location are illustrated.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Overall immune cell composition stratified by viral load group for Case 3 is summarised\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/5ce7517298c2b88ddb9051d8.png"},{"id":104876773,"identity":"52ad56e5-2690-4527-9dcf-1701b664a3ee","added_by":"auto","created_at":"2026-03-18 08:43:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eExpression-based pathway network across all viral load conditions.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eEach node represents a biological pathway, coloured by the viral load group in which it shows highest relative. Edges connect pathways with highly similar expression patterns across conditions, defined by Pearson correlation (r \u0026gt; 0.8). Node positions reflect functional proximity derived from graph layout, forming a pseudotemporal map of immune response dynamic, including all cases \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(A.)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, and only Case 3 \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B.)\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/cb681092b19c8c326492eda9.png"},{"id":104876636,"identity":"ec1f0b9e-c527-4906-8598-9fe9ba5d2ee7","added_by":"auto","created_at":"2026-03-18 08:43:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDifferential gene expression in BoDV-1-infected brain regions across viral load stages and in comparison, to non-infected control tissue.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eVolcano plots display pairwise comparisons between brain regions with differing BoDV-1 viral loads and control tissue. Genes significantly upregulated (red) or downregulated (blue) are highlighted according to significance thresholds (p \u0026lt; 0.05 and p \u0026lt; 0.01)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/d71702e2a15afb8512605e21.png"},{"id":104876667,"identity":"eea595e6-6b5e-43ee-a3a3-69d5857fb22b","added_by":"auto","created_at":"2026-03-18 08:43:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eKEGG Enrichment Analysis with GSEA Summary.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eGSEA using KEGG pathways was performed across BoDV-1 viral load groups.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eDot size represents significance of pathway enrichment, and the NES the strength and direction of enrichment. KEGG-defined host antiviral response pathways capture conserved immune programs that are commonly activated during viral infections and autoimmune inflammatory diseases, reflecting shared antiviral mechanisms. A custom gene set comprising antiviral and neuronal stress-associated genes described in herpesvirus (HSV-1, EBV, VZV), rabies, TBE, and measles infections, as well as inflammatory and demyelinating conditions including PML, MS, ADEM, and RE, was included to enable comparative analyses across neurotropic viral and immune-mediated CNS pathologies\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/f053fcf8563da8acb60ac111.png"},{"id":104876883,"identity":"82e4c6c1-66be-4846-9afd-f933d704c455","added_by":"auto","created_at":"2026-03-18 08:43:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18559213,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/16c06d90-6c3b-4e97-9242-cbad97cc6505.pdf"},{"id":104876657,"identity":"d1f878d0-bd8c-4cdf-80d5-976141e7741d","added_by":"auto","created_at":"2026-03-18 08:43:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3912287,"visible":true,"origin":"","legend":"","description":"","filename":"SUBMITJungbackAdditionalfileActaNeuropathologicaCommunications.docx","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/c0d6c87929c2527c669604b3.docx"},{"id":104876669,"identity":"c269e59e-300c-4b05-97b0-23fe805dc8aa","added_by":"auto","created_at":"2026-03-18 08:43:19","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15063534,"visible":true,"origin":"","legend":"","description":"","filename":"SUBMITJungbackFiguresActaNeuropathologicaCommunications.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9072167/v1/dc0579212ebf237356a68789.pptx"}],"financialInterests":"Competing interest reported. FLS receives funding from the Deutsche Forschungsgemeinschaft (504757758), support from the Bavarian State Ministry of Health, Care, and Prevention (Zoonotic Bornavirus Focalpoint Bavaria [ZooBoFo]), and has received honorarium for a scientific presentation on bornavirus encephalitis at Ingolstadt Hospital (Ingolstadt, Germany). FLS and PA were supported by the Kurscheid-Hühnlein-Stiftung.","formattedTitle":"Multimodal Profiling of Immune Responses Reveals Innate-Adaptive Immune Imbalance in Human Bornavirus Encephalitis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eHuman bornavirus encephalitis\u003c/strong\u003e (BVE) is a rare but mostly fatal disease. While the main causative pathogen, the Borna disease virus 1 (BoDV-1), has been known to infect animals for decades, the first confirmed human cases were only identified in 2018 [17]. Since then, BoDV-1 has been recognised as a significant zoonotic pathogen in the endemic areas in Southern and Eastern Germany [3]. Approximately five to ten new human cases are confirmed each year, with a cumulative total of around 50 known cases to date [13].Clinically, BoDV-1 infection often begins with nonspecific, flu-like symptoms, followed by rapid neurological deterioration with altered consciousness, seizures, and motor deficits. In most cases, the disease progresses swiftly, leading to death within a few weeks after symptom onset\u0026nbsp;[6, 13, 23]. The combination of high case fatality, rapid progression, and diagnostic challenges underscore the urgent need for increased clinical awareness and further therapeutic research, while also positioning BVE as valuable model for studying the pathogenesis of highly neurotropic viruses in general.\u003c/p\u003e\n\u003cp\u003eOur initial neuropathological investigations described the first histological characteristics of BVE in six human autopsy cases. The viral distribution pattern showed prominent involvement of the basal ganglia with histological definition as a sclerosing panencephalitis with lymphocytic infiltration, microglial nodules, and intranuclear eosinophilic inclusions [7]. Subsequent findings revealed the presence of BoDV-1 in endothelial cells, observed under certain conditions and linked to vascular injury caused by hypoxic stress [8]. Finck\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e extended the understanding of BVE by correlating magnetic resonance imaging (MRI) findings with histopathological changes in a separate set of cases. They identified a reproducible pattern of radiological changes, beginning in the caudate nucleus and insular cortex, followed by involvement of the limbic system and brainstem [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough the exact immunopathological mechanisms remain unclear to date, evidence suggests that BoDV-1 is non-cytolytic, with tissue injury primarily immune-mediated, involving CD4⁺ and CD8⁺ T cells, as well as reactive astrocytes, which are increasingly recognised as active contributors to disease progression [11, 14]. In selected cases, immunosuppressive treatment has been associated with prolonged survival, underscoring the potential role of immune modulation in disease management [6, 14]. Serum and cerebrospinal fluid analysis from ten individuals with BVE demonstrated a predominantly proinflammatory cytokine profile, indicating sustained recruitment of immune cells. Such an inflammatory milieu may also disrupt astrocyte function, potentially resulting in neuronal excitotoxicity [14].\u003c/p\u003e\n\u003cp\u003eAlthough individual immunopathological features of BVE have been described, the spatial viral distribution within the human brain and the neuroimmune responses remain insufficiently characterised. To address these gaps, we systematically analysed BoDV-1 dissemination in fully embedded, anatomically preserved brain cross-sections, and performed in-depth, transcriptome-based profiling of host neuroimmune responses. We hypothesised that BoDV-1 infection induces a distinct neuroinflammatory response shaped by viral tropism and local tissue context. Beyond advancing the understanding of BVE pathogenesis, our findings support the use of BVE as model to study host-pathogen interactions in infections caused by neurotropic viruses.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003eMaterial\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBrain autopsy material from four individuals (ages 39-71 years; median 68.5 years; all female) who died of BVE between 2022 and 2024 was included.\u0026nbsp;Cases 3 and 4 were previously described [2, 23], whereas Cases 1 and 2 have not been published yet.\u003c/p\u003e\n\u003cp\u003eFor Cases 1 and 2, complete sagittal sections of the right hemisphere and complete coronal sections of the left hemisphere were prepared. Case 3 included a right sagittal section; Case 4 a coronal section through both hemispheres to assess viral distribution symmetry. Cross-sections were subdivided into approximately equally sized blocks and embedded into capsules, allowing for later reconstruction of the whole-brain slices. In total, 157 formalin-fixed, paraffin-embedded (FFPE) blocks were included in the analyses (median 37 blocks per case; range 31 to 52). Control tissue from a 64-year-old woman who died of toxic cardiocirculatory failure in the setting of advanced metastatic breast carcinoma and pleural effusion included two FFPE blocks (left thalamus and frontal cortex).\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the ethics committee of the Ludwig-Maximilians-University ethics committee, responsible for Augsburg University Hospital (approval no. 23-0267).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eGeneral Study Design and Workflow\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs outlined in Figure 1, all FFPE blocks were stained with hematoxylin and eosin (HE) and BoDV-1 immunohistochemistry (IHC) using automated staining systems (HE: Tissue-Tek Prisma, Sakura, Japan; IHC: Leica Bond RX, Leica Biosystems, Germany). In Case 4, additional immunostainings for T cell marker CD3, B cell marker CD20, microglia marker Iba-1, and astrocytic marker GFAP were performed. A total of 450 slides were prepared and digitised (scanner Pannoramic II, 3DHISTECH, Hungary).\u003c/p\u003e\n\u003cp\u003eSagittal and coronary cross-sections were digitally reconstructed. Based on BoDV-1 immunoreactivity, automated signal quantification analysis using the QuPath software [1] was performed, generating a histopathological (H-)Score for each FFPE block. Additionally, CellQuant software (3DHISTECH, Hungary) was used for enhanced visualisation of BoDV-1 signal distribution. RNA was separately extracted from all blocks to determine BoDV-1 viral loads. For transcriptomic profiling, 12 FFPE blocks per case (covering low, medium, and high viral loads; total: 50 samples, incl. two controls) were analysed using the nCounter Sprint Profiler (NanoString/Bruker, United States) to characterise immunological gene expression profiles. The resulting data were subjected to statistical analysis using custom Python (Python Software Foundation, United States) workflows. Details on all methods are provided in the \u003cem\u003eSupplementary Material.\u003c/em\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003eRegional BoDV-1 Viral Load Profiling Reveals Marked Interindividual Variability but Consistent Hotspots in Deep Brain Structures\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCoronal and sagittal brain sections were analysed using three complementary approaches (Fig. 2A): BoDV-1 immunoreactivity was assessed via CellQuant-based digital visualisation and semi-quantitative H-Scoring using QuPath (see\u003cem\u003e\u0026nbsp;Supplementary Material Table 1\u003c/em\u003e). In parallel, BoDV-1 RNA levels were determined by real-time quantitative polymerase chain reaction (RT-qPCR) across all FFPE blocks. For the standardised anatomical assignment of tissue blocks see \u003cem\u003eSupplementary Material Figure\u0026nbsp;1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eViral loads were subsequently classified into low, medium, and high categories, first within each individual case and then across all cases. Due to marked interindividual variability, distinct thresholds were applied in each context.\u003c/p\u003e\n\u003cp\u003eMean viral loads differed interindividually by several orders of magnitude with ranges from 170.37 copies(c)/ng RNA in Case 4 to 462 711.72 c/ng RNA in Case 3. Case 1 (94 998.83 c/ng RNA) and Case 2 (9 343.39 c/ng RNA) showed intermediate values. Across all cases, FFPE blocks containing the basal ganglia, hippocampus, thalamus, and brainstem consistently showed higher viral loads. This regional pattern was mirrored by immunohistochemistry: H-Score analysis revealed the highest mean score in Case 3 (54.35), and the lowest in Case 4 (6.11), with intermediate values in Cases 1 (36.23) and 2 (46.77; \u003cem\u003eSupplementary Material Tables 2 and 3\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eBoDV-1 RNA levels and antigen detection showed weak but statistically significant positive correlations (Pearson\u0026rsquo;s \u0026rho; = 0.317, p \u0026lt; 0.001; Spearman\u0026rsquo;s \u0026rho; = 0.244, p = 0.002), consistent with a parallel distribution of viral RNA and immunoreactivity for BoDV-1 nucleoprotein.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eBilateral Viral Load Comparison Shows Symmetric Distribution in Case 4\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn Case 4, paired measurements of viral load revealed a highly symmetric distribution between both hemispheres. Pearson correlation analysis demonstrated a strong positive linear correlation (\u0026rho; = 0.951, p \u0026lt; 0.001) between the respective FFPE blocks, supporting a symmetric viral spread. This was corroborated by a paired t-test, which showed no significant difference between hemispheres (t = -0.974, p = 0.3480).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eCorrelation Analysis Identifies Coordinated T Cell, Microglial, and Astrocytic Responses to BoDV-1 RNA Levels\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor Case 4, an intra-case correlation analysis was performed to explore associations between BoDV-1 levels and cellular immune markers. Spearman correlation coefficients were calculated for BoDV-1 RNA levels, and the expression levels of CD3 (T cells), CD20 (B cells), Iba-1 (microglia), and GFAP (astrocytes). The analysis revealed several significant correlations (\u003cem\u003eSupplementary Material Figure 2\u003c/em\u003e). BoDV-1 RNA levels showed a moderate positive correlation with CD3-positive cells (\u0026rho; = 0.587, p \u0026lt; 0.001) and correlated moderately with Iba-1 (\u0026rho; = 0.502, p = 0.003), and GFAP (\u0026rho; = 0.406, p = 0.019), according to the classification by Schober \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e[19]\u003cem\u003e.\u003c/em\u003e Additionally, all pairwise combinations of CD3, Iba-1, and GFAP showed significant positive correlations (CD3/GFAP: \u0026rho; = 0.493, p = 0.003; CD3/Iba-1: \u0026rho; = 0.403, p = 0.018; Iba-1/GFAP: \u0026rho;= 0.480, p = 0.004).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eTranscriptomic Immune Cell Mapping Suggests Viral Load-dependent Modulation of Immune and Glial Populations\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo systematically assess the relationship between viral burden and immune activation, all tissue samples were stratified into three groups (BoDV-1\u003csub\u003ehigh\u003c/sub\u003e,\u0026nbsp;BoDV-1\u003csub\u003emedium\u003c/sub\u003e, BoDV-1\u003csub\u003elow\u003c/sub\u003e) based on viral RNA copy numbers (thresholds listed in Fig. 2A). A non-infected control served as reference. In total,\u0026nbsp;50 tissue samples were selected for transcriptome-based profiling using the nCounter platform, providing high-resolution insights into the immune microenvironment across varying viral loads in different brain areas.\u0026nbsp;Immune cell composition was inferred by transcriptomic deconvolution using cell-type-specific gene signatures and pathways, covering not only brain-specific cell types such as astrocytes and microglia, but also immune cells attracted to the site of inflammation. The aim was to determine whether distinct immune cell subsets are differentially enriched across varying levels of viral loads.\u003c/p\u003e\n\u003cp\u003eThe marked interindividual differences in viral burden necessitated an additional intra-case evaluation for Case 3, which exhibited by far the highest viral RNA levels. As shown in Figure 2A, the thresholds for viral load categories were accordingly adapted.\u003c/p\u003e\n\u003cp\u003eAcross all BoDV-1-infected brain samples, a clear viral load-dependent modulation of immune cell populations was observed (\u003cem\u003eSupplementary Material Figure 3A\u003c/em\u003e). Dendritic cells emerged as the most abundant population under all viral load groups. Macrophages were the second most prominent population, which seems consistent with their role in phagocytic clearance and cytokine release. Basophils and neutrophils showed moderate enrichment, which likely reflects a secondary influx in response to tissue damage and inflammation. Other immune subsets, including monocytes, natural killer (NK) cells, and T cells, exhibited a similar abundance in response. Plasma cells, eosinophils, B cells, mast cells, and myeloid-derived suppressor cells, were only sparsely detected, indicating a limited role in the dominant inflammatory response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough T cells were not the most dominant immune population, they play a crucial role in the immune response to BoDV-1 infection, and, exhibiting cuffing around blood vessels and tissue infiltration are a histopathological hallmark of encephalitis. A more detailed analysis of T cell subsets revealed that CD8⁺ T cells were the most abundant across all BoDV-1 viral load groups, followed by CD4⁺ and memory T cells (\u003cem\u003eSupplementary Material Figure 3B\u003c/em\u003e). The distribution followed a similar pattern, with the low groups exhibiting slightly higher values than the medium viral load groups, particularly for the CD8⁺ T cells. For CD4⁺ and memory T cells, the distribution was relatively even across all groups.\u003c/p\u003e\n\u003cp\u003eA closer look at the astrocyte and microglia subsets revealed a similar distribution pattern with predomination of the disease-associated subtype (\u003cem\u003eSupplementary Material Figure 3 C/D\u003c/em\u003e). In astrocytes, this was followed by the metabolic and reactive subtypes, while in microglia, the interferon-\u0026gamma;-induced subtype ranked second. These findings suggest a strong association between the viral load and the activation of specific immune and glial cell subsets.\u003c/p\u003e\n\u003cp\u003eTo minimise variability between individuals and confirm the findings under more controlled conditions, a within-case analysis was repeated for Case 3. Notably, the results closely resembled the patterns seen across all BoDV-1 viral load groups, supporting the homogeneity of the observed immune profile. Dendritic cells again emerged as the predominant population, irrespective of local viral load (Fig. 2B/C), followed by macrophages. Neutrophils and basophils were also repeatedly detected, indicating innate immune recruitment in response to infection or tissue damage. Although monocytes, NK cells, and T cells appeared less frequently, they were consistently present (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 4A\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eA more detailed analysis of T cells, astrocytes, and microglia confirmed this trend: CD8⁺ T cells were again by far the most dominant subset and their relative abundance slightly decreased in BoDV-1\u003csub\u003emedium\u003c/sub\u003e regions (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 4B\u003c/em\u003e), accompanied by a marked accumulation of disease-associated astrocytes (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 4C\u003c/em\u003e) and microglia (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 4D\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ePathway and Pseudotime Analysis Reveal Load-dependent Innate Activation and Limited Adaptive Immune Engagement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAcross all BoDV-1-infected brain samples, transcriptomic patterns across different viral load levels were dominated by innate immune signalling pathways, with progressive upregulation of interferon-stimulated genes (ISGs), antigen presentation, protein synthesis, and stress-response pathways (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 5A\u003c/em\u003e) indicating dose-dependent activation of type I interferon signalling via pattern recognition receptors, a defining feature of acute antiviral immunity [18].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe within-case analysis of Case 3 (\u003cem\u003eSupplementary Material\u0026nbsp;Figure 5B\u003c/em\u003e) mirrored these patterns, showing the same ISG- and antigen-presentation-driven response with similar distribution across viral load groups, despite minor shifts in the relative ranking of pathways.\u003c/p\u003e\n\u003cp\u003eThe pathway-level expression network revealed a structured immune response aligned with viral load (Fig. 3A). In BoDV-1 encephalitis, pathway clustering across pseudotime reflected a progression from early cellular maintenance to advanced immune activation. Low viral load tissue was dominated by Notch signalling, which occupied an isolated position at the early end of the pseudotime axis. Medium viral load samples were associated with DNA repair, protein synthesis, signal transduction, angiogenesis, and Wnt signalling, forming a central cluster indicative of early-to-intermediate cellular activation. In contrast, high viral load samples displayed dense clustering of stress- and immune-related pathways.\u003c/p\u003e\n\u003cp\u003eOf note, adaptive immune pathways were located at the interface between medium and high viral load clusters, suggesting a transitional or delayed engagement of effector responses. Control samples did not form a dominant cluster, underscoring their limited role in the overall immune activation profile.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Case 3, low and high viral load samples dominated the expression landscape (Fig. 3B). Low viral load was associated with early response pathways such as DNA repair,\u0026nbsp;adaptive immunity, and\u0026nbsp;angiogenesis, whereas high viral load corresponded to robust activation of\u0026nbsp;innate immunity, oxidative stress, inflammation, and immune checkpoints. Notably, adaptive immune signalling\u0026nbsp;formed no coherent cluster, suggesting a limited transition to effective T cell-mediated responses despite persistent viral presence.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eGene Expression Analysis Shows Threshold-dependent Activation of Innate and Inflammatory Pathways\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptomic profiling across all four cases revealed viral load-dependent immune signatures. Differential gene expression was most pronounced in comparisons involving BoDV-1\u003csub\u003ehigh\u003c/sub\u003e samples (vs. BoDV-1\u003csub\u003emedium\u003c/sub\u003e and vs. BoDV-1\u003csub\u003elow\u003c/sub\u003e; Fig. 4), supporting that substantial immune activation occurs only above a critical viral threshold. \u0026nbsp;In BoDV-1\u003csub\u003ehigh\u003c/sub\u003e tissue, the marked upregulation of CD68, IL1B, and CCL3 indicated pronounced microglial activation and proinflammatory signalling. The consistent increase in CASP8 expression further suggested an engagement of inflammatory cell death pathways under high viral load conditions. Compared to uninfected controls, BoDV-1\u003csub\u003ehigh\u003c/sub\u003e samples further showed increased expression of CCR5, supporting an enhanced chemokine-mediated immune cell recruitment to the CNS [21]. Interestingly, CXCL9, a pro-inflammatory chemokine involved in T cell recruitment, was significantly underexpressed in BoDV-1\u003csub\u003emedium\u003c/sub\u003e compared to BoDV-1\u003csub\u003elow\u003c/sub\u003e samples (Fig. 4B).\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;Case 3, BoDV-1\u003csub\u003ehigh\u003c/sub\u003e samples exhibited pronounced transcriptional changes consistent with strong antiviral immune activation (\u003cem\u003eSupplementary Material Figure 6\u003c/em\u003e). Most significantly upregulated genes included\u0026nbsp;CD68 (activated microglia/macrophages), TLR2 (sensor of viral ligands),\u0026nbsp;OAS1 (interferon-stimulated antiviral effector), and CXCL10 (T cell recruiting chemokine), reflecting coordinated activation of innate immune sensors and CNS-resident effector cells. Upregulation of\u0026nbsp;FCGR3 supports a role for antibody-dependent innate mechanisms [10].\u003c/p\u003e\n\u003cp\u003eComparison between medium and low viral load revealed only minor changes. Compared to controls, BoDV-1\u003csub\u003ehigh\u003c/sub\u003e samples showed increased CASP3 and CCR5, indicating enhanced cytotoxicity and leukocyte recruitment [21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ePathway-based Gene Set Analysis Reveals Upregulation of Non-lytic Viral and Autoimmune-like Signatures\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA Kyoto Encyclopedia of Genes and Genomes (KEGG)-based gene set enrichment analysis (GSEA), including neurotropic, inflammatory, and non-pathogen-associated encephalitic reference sets, revealed a structured and category-specific transcriptional response across BoDV-1 viral load groups (Fig. 5). Compared to control tissue, the signatures for herpes simplex virus 1 (HSV-1), measles and Epstein-Barr virus were downregulated across all groups. In contrast, the signatures for the neurotropic tick-borne virus, rabies virus (RV), varicella-zoster virus and JC-virus (causative agent of progressive multifocal leukoencephalopathy) were uniformly upregulated. Interestingly, signatures for autoimmune diseases, such as multiple sclerosis and acute disseminated encephalomyelitis also showedupregulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBVE is an emerging, severe and mostly fatal inflammatory disease of the central nervous system without any established treatment regimen. Neuropathological studies describe a lymphocyte-rich panencephalitis with widespread lesions showing intranuclear inclusions in neurons and glial cells, microglial nodule formation, astrocyte activation, and severe tissue damage primarily driven by a strong proinflammatory immune response. Although involvement of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T cells, microglial and astrocytic activation, and proinflammatory cytokine production has been described [7, 11, 14], \u0026nbsp;the associated neuroimmune responses in the human brain remain poorly understood. This study aimed to address this gap through anatomically resolved viral mapping and transcriptome-based immune profiling.\u003c/p\u003e\n\u003cp\u003eOur results show that BoDV-1 viral loads varied markedly between individuals, likely reflecting differences in treatment regimens rather than purely biological factors. High viral burdens in the basal ganglia, hippocampus, thalamus, and brainstem are consistent with previous reports [4, 5, 7]. Correlation between BoDV-1 RNA levels and immune cell and glial markers suggest an association of viral replication with T cell infiltration and reactive activation of microglia and astrocytes, features prominently observed in neuropathological examinations of BVE cases [7, 14]. Immune cell populations in BoDV-1-infected brain samples displayed distinct activation patterns linked to viral load levels. Local dendritic cells together with attracted macrophages, neutrophils, and basophils were the most abundant immune subsets, while CD8⁺ T lymphocytes dominated the adaptive compartment, consistent with Rauch \u003cem\u003eet al.\u003c/em\u003e [14]. Although a moderate positive correlation between viral RNA levels and CD3⁺ T cell abundance was observed, stratification by viral load indicates a more nuanced, potentially stage-specific regulation of cytotoxic T cell responses.\u003c/p\u003e\n\u003cp\u003eThe activation of ISGs, antigen presentation, protein synthesis, and oxidative stress pathways reflects a robust antiviral and cellular stress response. Notably, our data suggest a relative underrepresentation of adaptive immune signalling, which may reflect either a temporal delay in adaptive responses or active suppression mechanisms. This aligns with established processes of immune regulation, including T cell exhaustion, checkpoint inhibition, and pathogen-driven immune evasion [24]. \u0026nbsp;Altogether, the findings indicate a tiered immune response in BVE, shifting from homeostatic and repair-associated states towards antiviral and stress-driven activation as viral loads increase. This constellation, characterised by a strong innate response alongside lower or suppressed adaptive immunity, may help explain why BoDV-1 is not cleared despite pronounced inflammation and severe tissue destruction, thereby likely contributing to the fatal disease course. Importantly, these data do not contradict the established concept of immune-mediated pathology in BVE, but highlight the complexity of immune regulation in advanced stages.\u003c/p\u003e\n\u003cp\u003eImmune activation occurred only above a critical viral threshold. Higher viral loads correlated with microglial activation, proinflammatory signalling, and cell death. Upregulation of CD68, IL1B, CCL3, CASP8, and CCR5, alongside downregulation of CXCL9 suggests a BoDV-1-driven immune environment that may limit antiviral T cell recruitment while stimulating microglia activation, potentially contributing to viral persistence. This finding highlights the need for a more detailed characterisation of microglia and their role during BoDV-1 infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen comparing with other neurotropic virus infections, such as HSV-1 and RV infection, overlapping immune features were observed in BVE. Both, BoDV-1 and HSV-1 infection show CD68 and CXCL10 upregulation [20, 22]. IL1B is also upregulated in RV-infected microglia [9]. However, notable differences emerged from our analyses. HSV-1 infection primarily leads to CCL2 and CCL5 upregulation [20], whereas BoDV-1 infection selectively induced CCL3. No human data on CXCL9 expression exist for HSV-1 or RV, with insights into its functional role during HSV-1 infection derived solely from animal models [25]. Additionally, BVE showed transcriptional activation of apoptosis-related genes (CASP8, CASP9, LC3A), while during HSV-1 infection, these are mainly regulated at the post-translational level; for RV, such data are lacking. These differences highlight virus-specific immunopathological mechanisms despite shared innate immune activation, suggesting that each virus engages distinct strategies to shape host responses, and influence disease outcome. This is corroborated by KEGG enrichment analyses, which showed little overlap between BoDV-1 infection and transcriptional programs characteristic of cell-lytic viruses like HSV-1. This may explain the differences despite shared activation of innate immune pathways including oxidative stress responses, autophagy regulation, and antigen presentation [12, 15, 16]. In contrast, gene signatures associated with non-lytic neurotropic viruses, such as RV and tick-borne encephalitis virus, were upregulated in BoDV-1 infection. Interestingly, gene sets related to autoimmune neuroinflammatory conditions were likewise enriched. This combination may indicate a uniquely dysregulated neuroimmune state, aligning with previous clinical and neuropathological observations, including corticosteroid-responsive inflammation and prominent lymphocytic involvement, as reported previously [5, 14, 23].\u003c/p\u003e\n\u003cp\u003eDespite the novelty of our investigations of the immunopathogenesis of BVE, some challenges and limitations have to be acknowledged. Human BVE is an extremely rare disease, thereby limiting the number of cases available for analysis. Moreover, heterogenous disease courses and treatment attempts, together with the terminal-stage autopsy material, differences in post-mortem interval, and tissue fixation time, limit generalisability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, even with these constraints, our data offer valuable insights into the immunopathogenesis of BVE. Collectively, the data indicate an imbalance characterised by robust activation of immune pathways in the presence of comparatively weak adaptive immune responses. Excessive innate immune activation may contribute to tissue injury and thereby limit effective immune control, potentially facilitating viral persistence. In line with this, pathway analyses showed little overlap with transcriptional programs of lytic viral infections but strong similarity to signatures of non-lytic neurotropic viruses and autoimmune neuroinflammation. Notably, animal models have demonstrated that impaired or immature immune responses can prevent disease manifestation, highlighting the critical role of host immune regulation in BoDV-1 encephalitis. Together, these findings suggest that adaptive immune mechanisms may contribute more substantially to BoDV-1 disease pathogenesis rather than the response being exclusively innate, and therefore warrant consideration in future therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003eEthics approval\u0026nbsp;\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was granted by the ethics committee of the Ludwig-Maximilians-University, responsible for Augsburg University Hospital (approval no. 23-0267).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eConsent to participate\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study used pseudonymised data and materials. Therefore, informed consent to participate was not required. Informed consent for autopsy was obtained from legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eConsent to publish\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data are fully anonymised, and no identifying information is included. Therefore, consent for publication was not required.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author.\u0026nbsp;The authors confirm that data supporting the findings of this study are also available in the Supplementary Material.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFLS receives funding from the Deutsche Forschungsgemeinschaft (504757758), support from the Bavarian State Ministry of Health, Care, and Prevention (Zoonotic Bornavirus Focalpoint Bavaria [ZooBoFo]), and has received honorarium for a scientific presentation on bornavirus encephalitis at Ingolstadt Hospital (Ingolstadt, Germany). FLS and PA were supported by the Kurscheid-Hühnlein-Stiftung. None of the funding bodies were involved in either the design of the study, nor the collection, analysis, and interpretation of data.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Mrs. Natalia Gerling and Mrs. Juliane Stephan for their excellent conduct of the laboratory work. Figure 1 and Figure 2B were created with BioRender.com. The BoDV-1 antibody Bo18 was kindly provided by the Friedrich-Loeffler-Institute.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eAuthor Contributions\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eManuscript Draft: NJ, FLS\u003c/p\u003e\n\u003cp\u003eStatistical Analysis: NJ, DH, MD\u003c/p\u003e\n\u003cp\u003eCreation of Illustrations: NJ, FLS\u003c/p\u003e\n\u003cp\u003eTissue analyses: BM, NJ, PG, FLS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: FLS\u003c/p\u003e\n\u003cp\u003eEditing, Review and Approval: PG, DH, MD, ZM, TP, TR, GR, AB, BM, KH, PA, DT\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBankhead P, Loughrey MB, Fern\u0026aacute;ndez JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HGet al (2017) QuPath: Open source software for digital pathology image analysis. Scientific Reports 7: 16878 Doi 10.1038/s41598-017-17204-5\u003c/li\u003e\n\u003cli\u003eBayas A, Menacher M, Lapa C, Tappe D, Maurer C, Liesche-Starnecker F, Schneider H, Naumann M (2024) 18fluorodeoxyglucose PET/CT as possible early diagnostic tool preceding MRI changes in Borna disease virus 1 encephalitis. Lancet (London, England) 403: 665\u0026ndash;666 Doi https://doi.org/10.1016/S0140-6736(24)00049-7\u003c/li\u003e\n\u003cli\u003eEbinger A, Santos PD, Pfaff F, D\u0026uuml;rrwald R, Kolodziejek J, Schlottau K, Ruf V, Liesche-Starnecker F, Ensser A, Korn Ket al (2024) Lethal Borna disease virus 1 infections of humans and animals - in-depth molecular epidemiology and phylogeography. Nat Commun 15: 7908 Doi 10.1038/s41467-024-52192-x\u003c/li\u003e\n\u003cli\u003eFinck T, Liesche-Starnecker F, Probst M, Bette S, Ruf V, Wendl C, Dorn F, Angstwurm K, Schlegel J, Zimmer Cet al (2020) Bornavirus Encephalitis Shows a Characteristic Magnetic Resonance Phenotype in Humans. Annals of neurology 88: 723\u0026ndash;735 Doi https://doi.org/10.1002/ana.25873\u003c/li\u003e\n\u003cli\u003eGrosse L, Lieft\u0026uuml;chter V, Vollmuth Y, Hoffmann F, Olivieri M, Reiter K, Tacke M, Heinen F, Borggraefe I, Osterman Aet al (2023) First detected geographical cluster of BoDV-1 encephalitis from same small village in two children: therapeutic considerations and epidemiological implications. Infection 51: 1383\u0026ndash;1398 Doi https://doi.org/10.1007/s15010-023-01998-w\u003c/li\u003e\n\u003cli\u003eJungb\u0026auml;ck N, Vollmuth Y, M\u0026ouml;gele T, Grochowski P, Schlegel J, Schaller T, M\u0026auml;rkl B, Herden C, Matiasek K, Tappe Det al (2025) Neuropathology, pathomechanism, and transmission in zoonotic Borna disease virus 1 infection: a systematic review. Lancet Infect Dis 25: e212-e222 Doi 10.1016/s1473-3099(24)00675-3\u003c/li\u003e\n\u003cli\u003eLiesche F, Ruf V, Zoubaa S, Kaletka G, Rosati M, Rubbenstroth D, Herden C, Goehring L, Wunderlich S, Wachter MFet al (2019) The neuropathology of fatal encephalomyelitis in human Borna virus infection. Acta neuropathologica 138: 653\u0026ndash;665 Doi https://doi.org/10.1007/s00401-019-02047-3\u003c/li\u003e\n\u003cli\u003eLiesche-Starnecker F, Schifferer M, Schlegel J, Vollmuth Y, Rubbenstroth D, Delbridge C, Gempt J, Lorenzl S, Schnurbus L, Misgeld Tet al (2022) Hemorrhagic lesion with detection of infected endothelial cells in human bornavirus encephalitis. Acta neuropathologica 144: 377\u0026ndash;379 Doi https://doi.org/10.1007/s00401-022-02442-3\u003c/li\u003e\n\u003cli\u003eLiu J, Li W, Yu D, Jin R, Hou H, Ling X, Kiflu AB, Wei X, Yang X, Li Xet al (2023) Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence. Viruses 15: Doi 10.3390/v15061223\u003c/li\u003e\n\u003cli\u003e Nimmerjahn F, Ravetch JV (2006) Fcgamma receptors: old friends and new family members. Immunity 24: 19-28 Doi 10.1016/j.immuni.2005.11.010\u003c/li\u003e\n\u003cli\u003e Nobach D, M\u0026uuml;ller J, Tappe D, Herden C (2020) Update on immunopathology of bornavirus infections in humans and animals. Adv Virus Res 107: 159-222 Doi 10.1016/bs.aivir.2020.06.004\u003c/li\u003e\n\u003cli\u003e Orvedahl A, Alexander D, Tall\u0026oacute;czy Z, Sun Q, Wei Y, Zhang W, Burns D, Leib DA, Levine B (2007) HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 1: 23-35 Doi 10.1016/j.chom.2006.12.001\u003c/li\u003e\n\u003cli\u003e P\u0026ouml;rtner K, Wilking H, Frank C, Stark K, Wunderlich S, Tappe D (2024) Clinical analysis of Bornavirus Encephalitis cases demonstrates a small time window for Etiological Diagnostics and treatment attempts, a large case series from Germany 1996\u0026ndash;2022. Infection: Doi 10.1007/s15010-024-02337-3\u003c/li\u003e\n\u003cli\u003e Rauch J, Steffen JF, Muntau B, Gisbrecht J, P\u0026ouml;rtner K, Herden C, Niller HH, Bauswein M, Rubbenstroth D, Mehlhoop Uet al (2022) Human Borna disease virus 1 encephalitis shows marked pro-inflammatory biomarker and tissue immunoactivation during the course of disease. 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Brain pathology (Zurich, Switzerland) 32: e13031 Doi https://doi.org/10.1111/bpa.13031\u003c/li\u003e\n\u003cli\u003e Sorce S, Myburgh R, Krause KH (2011) The chemokine receptor CCR5 in the central nervous system. Prog Neurobiol 93: 297-311 Doi 10.1016/j.pneurobio.2010.12.003\u003c/li\u003e\n\u003cli\u003e Uyar O, Laflamme N, Piret J, Venable M-C, Carbonneau J, Zarrouk K, Rivest S, Boivin G (2020) An Early Microglial Response Is Needed To Efficiently Control Herpes Simplex Virus Encephalitis. Journal of Virology 94: 10.1128/jvi.01428-01420 Doi doi:10.1128/jvi.01428-20\u003c/li\u003e\n\u003cli\u003e Vollmuth Y, Jungb\u0026auml;ck N, M\u0026ouml;gele T, Schmidt-Graf F, Wunderlich S, Schimmel M, Rothe C, Stark L, Schlegel J, Rieder Get al (2024) Comparative Study of Virus and Lymphocyte Distribution with clinical Data suggests early high dose Immunosuppression as potential Key Factor for the Therapy of Patients with BoDV-1 Infection. 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Cell Immunol 243: 83-89 Doi 10.1016/j.cellimm.2007.01.001\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BoDV-1, encephalitis, neurotropic virus, immunopathogenesis, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-9072167/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9072167/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Human bornavirus encephalitis (BVE) is a rare, emerging and fatal zoonotic disease mainly caused by the Borna disease virus 1 (BoDV-1), a non-cytolytic RNA virus. Despite increasing recognition, the immunopathogenesis of human BoDV-1 infection remains unexplored. Complete coronary and sagittal brain sections from four fatal BoDV-1 cases were analysed using digitised immunohistochemistry to quantify viral distribution and tissue responses. Transcriptome-based analyses characterised local immune cell profiles in relation to viral loads measured by RT-qPCR. BoDV-1 viral loads varied substantially between cases but showed region-specific enrichment in the basal ganglia and hippocampus, correlating with lymphocyte presence and reactive microglia and astrocytes. Immune cell deconvolution revealed viral load-dependent modulation dominated by innate immune and glial populations, including metabolic and reactive astrocyte states, IFNγ-responsive microglia, and dendritic cells, macrophages, neutrophils, basophils, and CD8⁺ T cells. This was accompanied by induction of interferon-stimulated genes, antigen presentation, protein synthesis, and oxidative stress pathways, with a transcriptional signature resembling non-lytic viral and autoimmune-like neuroinflammatory conditions rather than lytic infections. These findings support a model of BoDV-1 encephalitis characterised by a dominant innate immune response and delayed adaptive immune engagement, which may contribute to ineffective viral clearance and extensive tissue damage.","manuscriptTitle":"Multimodal Profiling of Immune Responses Reveals Innate-Adaptive Immune Imbalance in Human Bornavirus Encephalitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:40:20","doi":"10.21203/rs.3.rs-9072167/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-04T17:25:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T14:53:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-28T18:44:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T01:34:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317677027712497136889395394857136953456","date":"2026-03-15T22:21:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321203393955739420233178838033771698162","date":"2026-03-15T21:04:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232231425004437806205271736996048203868","date":"2026-03-14T04:57:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T15:46:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-12T17:52:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-12T02:11:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2026-03-09T11:11:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d1740a2-a685-4465-bea9-7a6aae5ed9c1","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-01T13:39:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:40:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9072167","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9072167","identity":"rs-9072167","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}