BoDV-1 RT-qPCR was performed according to a previously published protocol [1]. Total RNA was extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany) on an EZ1 Advanced XL system (Qiagen), according to the manufacturer's instructions. 5 µL eluate was used for one-step reverse transcription and amplification in a total reaction volume of 30 µL using the TaqPath™ 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific, Waltham, MA, USA). BoDV-1 RNA was detected using two real-time RT-qPCR assays: primers and probe of mix 1 targeting the BoDV-1 X/phosphoprotein (P) gene region and primers and probes of mix 6 targeting the BoDV-1 matrix (M)/glycoprotein (G) gene region [1]. RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). In vitro-transcribed RNA molecules were used as standards for quantification, generated as previously described for a different detection assay [13]. For extraction and inhibition control, samples were spiked with a defined amount of MS2 bacteriophages and a MS2 phage RT-qPCR was performed [14]. All samples were tested in two replicates.

10 µm-thick sections were cut from a formalin-fixed paraffin-embedded (FFPE) brain tissue block and underwent deparaffinization (Deparaffinization Solution, Qiagen, Hilden, Germany), followed by RNA extraction using the RecoverAll™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer´s instructions. Quality and integrity of the extracted RNA were confirmed using the Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific) and the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). A total amount of 50 ng extracted RNA was subjected to viral metagenomic sequencing on a NextSeq 2000 platform using the 200-cycle (2 × 100 bp paired-end) NextSeq 2000 P2 Reagent Kit (Illumina, San Diego, CA, USA). The raw data generated were cleaned from low-quality reads and polyclonal sequences were removed. The viral genome was obtained using Geneious Prime (Biomatters, Auckland, New Zealand). The nucleotide sequence of the BoDV-1 genome generated in this study has been deposited in GenBank (accession no. PP272805).

A BoDV-1 indirect immunofluorescence assay (iIFA) was performed as previously described [1, 15]. Vero cells persistently infected with BoDV-1 (isolate Regensburg 2020) served for the detection of BoDV-1-reactive IgG antibodies. A secondary Cy-3-conjugated polyclonal rabbit anti-human-IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) was used in a dilution of 1:200.

A BoDV-1 IgM ELISA system was performed as previously described elsewhere [9]. Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated with either recombinant BoDV-1 N, X or P protein. For all samples, a pre-incubation with RF Absorbent (Virion/Serion, Würzburg, Germany) was performed according the manufacturer´s protocol; all samples (serum, CSF) were used in a dilution of 1:100. A secondary polyclonal rabbit anti-human IgM/HRP antibody (Agilent) was used in a dilution of 1:3,000. To control for inter-assay variability, optical density (OD) values were normalised to pre-characterised positive control samples of patients with confirmed BoDV-1 infection.

The BoDV-1 ELISpot was performed exactly as previously described elsewhere [12]. Peripheral blood mononuclear cells (PBMC) were isolated from lithium-heparin blood (2 × 7.5 mL) within 24 h after the sample was obtained (pre-analytical storage at room temperature). BoDV-1-derived 11-aa-overlapping 15-mer peptide pools spanning full-length viral nucleoprotein (N), accessory X protein or phosphoprotein (P) (peptides and elephants, Hennigsdorf, Germany) were used to stimulate BoDV-1-specific T cells within the fraction of PBMC. Stimulation with phytohemagglutinin (PHA) solution (T-SPOT.TB Kit, Oxford Immunotec, Abingdon, UK) served as positive control.

The retrospective examination of clinical samples of patients with encephalitis for the detection of new viruses such as BoDV-1 was approved by the Ethics Committee of the Faculty for Medicine, University of Regensburg, Regensburg, Germany (Reference Number: 18–1248-101).

Graphs were created using GraphPad Prism version 10.1.0 (GraphPad Software, San Diego, CA, USA).