Long-term elevation of complement factors in cerebrospinal fluid of patients with Borna disease virus 1 (BoDV-1) encephalitis.

BACKGROUND: Borna disease virus 1 (BoDV-1) causes rare but severe zoonotic infections in humans, presenting as severe encephalitis. The case-fatality risk is very high and no effective countermeasures have been established so far. An immunopathology is presumed, while data on immune responses in humans are limited. Evidence of a role of the complement system in various neurological disorders and central nervous viral infections is increasing and specific inhibitors are available as therapeutic options. METHODS: In this study, we investigated factors of the complement system in the cerebrospinal fluid (CSF) of patients with BoDV-1 infections (n = 17) in comparison to non-inflammatory control CSF samples (n = 11), using a bead-based multiplex assay. In addition, immunohistochemistry was performed using post-mortem brain tissue samples. RESULTS: We found an intrathecal elevation of complement factors of all complement pathways and an active cascade during human BoDV-1 infections. The increase of certain complement factors such as C1q was persistent and C3 complement deposits were detected in post-mortem brain sections. Intrathecal complement levels were negatively correlated with survival. CONCLUSION: Further investigations are warranted to clarify, whether targeting the complement cascade by specific inhibitors might be beneficial for patients suffering from severe BoDV-1 encephalitis.

Detection of virus-specific T cells via ELISpot corroborates early diagnosis in human Borna disease virus 1 (BoDV-1) encephalitis.

BACKGROUND: Within endemic regions in southern and eastern Germany, Borna disease virus 1 (BoDV-1) causes rare zoonotic spill-over infections in humans, leading to encephalitis with a high case-fatality risk. So far, intra-vitam diagnosis has mainly been based on RT-qPCR from cerebrospinal fluid (CSF) and serology, both being associated with diagnostic challenges. Whilst low RNA copy numbers in CSF limit the sensitivity of RT-qPCR from this material, seroconversion often occurs late during the course of the disease. CASE PRESENTATION: Here, we report the new case of a 40 - 50 year-old patient in whom the detection of virus-specific T cells via ELISpot corroborated the diagnosis of BoDV-1 infection. The patient showed a typical course of the disease with prodromal symptoms like fever and headaches 2.5 weeks prior to hospital admission, required mechanical ventilation from day three after hospitalisation and remained in deep coma until death ten days after admission. RESULTS: Infection was first detected by positive RT-qPCR from a CSF sample drawn four days after admission (viral load 890 copies/mL). A positive ELISpot result was obtained from peripheral blood collected on day seven, when virus-specific IgG antibodies were not detectable in serum, possibly due to previous immune adsorption for suspected autoimmune-mediated encephalitis. CONCLUSION: This case demonstrates that BoDV-1 ELISpot serves as additional diagnostic tool even in the first week after hospitalisation of patients with BoDV-1 encephalitis.

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).

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.

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.

Human Borna disease virus 1 encephalitis shows marked pro-inflammatory biomarker and tissue immunoactivation during the course of disease.

Human Borna disease virus 1 (BoDV-1) encephalitis is a severe emerging disease with a very high case-fatality rate. While the clinical disease, case definitions, diagnostic algorithms and neuropathology have been described, very little is known about the immunological processes of human BoDV-1 encephalitis. Here, we analyzed serum and cerebrospinal fluid (CSF) samples from 10 patients with fatal BoDV-1 encephalitis for changes of different cytokines, chemokines, growth factors and other biomarkers over time. From one of these individuals, also autoptic formalin-fixed brain tissue was analyzed for the expression of inflammatory biomarkers by mRNA levels and immunostaining; in a further patient, only formalin-fixed brain tissue was available and examined in addition. A marked and increasing immune activation from the initial phase to the last phase of acute BoDV-1 encephalitis is shown in serum and CSF, characterized by cytokine concentration changes (IFNγ, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-13, IL-18, TGF-ß1) with a predominantly pro-inflammatory pattern over time. IFNγ production was demonstrated in endothelial cells, astrocytes and microglia, IL-6 in activated microglia, and TGF-ß1 in endothelial cells, activated astrocytes and microglia. This was paralleled by an increase of chemokines (CCL-2, CCL-5, CXCL-10, IL-8) to attract immune cells to the site of infection, contributing to inflammation and tissue damage. Pathologically low growth factor levels (BDNF, ß-NGF, PDGF) were seen. Changed levels of arginase and sTREM further fostered the pro-inflammatory state. This dysbalanced, pro-inflammatory state likely contributes importantly to the fatal outcome of human BoDV-1 encephalitis, and might be a key target for possible treatment attempts.

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.

Increased neutralization of SARS-CoV-2 Delta variant after heterologous ChAdOx1 nCoV-19/BNT162b2 versus homologous BNT162b2 vaccination.

Heterologous SARS-CoV-2 vaccine approaches with a second mRNA-based vaccine have been favored in the recommendations of many countries over homologous vector-based ChAdOx1 nCoV-19 vaccination after reports of thromboembolic events and lower efficacy of this regimen. In the middle of 2021, the SARS-CoV-2 Delta variant of concern (VoC) has become predominant in many countries worldwide. Data addressing the neutralization capacity of a heterologous ChAdOx1 nCoV-19/mRNA-based vaccination approach against the Delta VoC in comparison to the widely used homologous mRNA-based vaccine regimen are limited. Here, we compare serological immune responses of a cohort of ChAdOx1 nCoV-19/BNT162b2-vaccinated participants with those of BNT162b2/BNT162b2 vaccinated ones and show that neutralization capacity against the Delta VoC is significantly increased in sera of ChAdOx1 nCoV-19/BNT162b2-vaccinated participants. This overall effect can be attributed to ChAdOx1 nCoV-19/BNT162b2-vaccinated women, especially those with more severe adverse effects leading to sick leave following second immunization.

Contribution of High Viral Loads, Detection of Viral Antigen and Seroconversion to Severe Acute Respiratory Syndrome Coronavirus 2 Infectivity.

BACKGROUND: From a public health perspective, effective containment strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should be balanced with individual liberties. METHODS: We collected 79 respiratory samples from 59 patients monitored in an outpatient center or in the intensive care unit of the University Hospital Regensburg. We analyzed viral load by quantitative real-time polymerase chain reaction, viral antigen by point-of-care assay, time since onset of symptoms, and the presence of SARS-CoV-2 immunoglobulin G (IgG) antibodies in the context of virus isolation from respiratory specimens. RESULTS: The odds ratio for virus isolation increased 1.9-fold for each log10 level of SARS-CoV-2 RNA and 7.4-fold with detection of viral antigen, while it decreased 6.3-fold beyond 10 days of symptoms and 20.0-fold with the presence of SARS-CoV-2 antibodies. The latter was confirmed for B.1.1.7 strains. The positive predictive value for virus isolation was 60.0% for viral loads >107 RNA copies/mL and 50.0% for the presence of viral antigen. Symptom onset before 10 days and seroconversion predicted lack of infectivity with negative predictive values of 93.8% and 96.0%. CONCLUSIONS: Our data support quarantining patients with high viral load and detection of viral antigen and lifting restrictive measures with increasing time to symptom onset and seroconversion. Delay of antibody formation may prolong infectivity.

Brief report: attitudes towards Covid-19 vaccination among hospital employees in a tertiary care university hospital in Germany in December 2020.

Coronavirus disease 2019 (Covid-19) vaccination is essential to fight the pandemic. Health care workers (HCWs) are prioritized to get vaccinated, yet uptake of recommended vaccinations is known to be low in this group. In a tertiary care university hospital with a high number of Covid-19 patients in intensive care, 59.5% of surveyed staff (N = 2454) were willing to get vaccinated, 21.4% were unsure and 18.7% refused. Vaccine hesitancy was higher in female, younger and healthy employees without contact to Covid-19 patients; nurses (53.3%) were much less willing to get vaccinated compared to physicians (82.7%).

Human T cells modulate myeloid-derived suppressor cells through a TNF-α-mediated mechanism.

Myeloid-derived suppressor cells (MDSC) represent an innate immune cell subset capable of suppressing T-cell responses in cancer and chronic inflammation. While the effect of MDSC on T cells has been defined thoroughly, the reciprocal impact of T cells on MDSC homeostasis remains poorly understood. Therefore, we comprehensively analyzed the effect of different T-cell subsets on the generation and survival of human MDSC. Using an in vitro MDSC generation assay, we demonstrate that unstimulated CD4+, but not CD8+ T cells, induce polymorphonuclear MDSC (PMN-MDSC) from CD33+ myeloid cells. This effect was dependent on direct cell-cell contact and required TNF-α signaling. Soluble TNF-α was dispensable for PMN-MDSC generation, suggesting that transmembrane TNF-α is involved in that trans-cellular process. Stimulated human CD3+ T cells delayed the apoptosis of PMN-MDSC, which was independent of TNF-α signaling or direct cell-cell contact, but was recapitulated by IL-2. Taken together, our study shows that human T cells modulate MDSC generation and survival through two distinct mechanisms and thereby fine-tune the homeostasis of human MDSC in a regulated manner.