Lethal Borna disease virus 1 infections of humans and animals – in-depth molecular epidemiology and phylogeography

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


Statistics
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Software and code
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Reporting on race, ethnicity, or other socially relevant groupings
Neither race, ethnicity, or other socially relevant groupings were recorded for the human patients.

Population characteristics
Sex, age, year of disease, duration of disease (from hospitalization to death), location of last residence, viral loads in brain samples and sequence of the infecting BoDV-1 were recorded for human patients.

Recruitment
Diagnostic material from BoDV-1-infected humans were submitted by diagnostic laboratories, pathologists or clinicians.The samples originated from diagnostic testing as well as from restrospective screening of archived material.All cases of laboratory-confirmed human BoDV-1 infection (following the definition of Eisermann et al., 2019) known to the authors were included in the study.Since direct virus detection (required for fulfilling the case definition criteria) may fail during intra vitam diagnosis, non-fatal BoDV-1 infection may be underrepresented among the studied cases.This has been discussed in the manuscript.

Ethics oversight
Ethical approval of the analysis of archived human samples was obtained from the local ethical commission of the Faculty for Medicine, University of Regensburg (ref. no. 18-1248-101), the Technical University Munich (577/19 S), the Ludwigs-Maximilians University Munich (23-0267) and the Medical Board of Hamburg (PV5616).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Life sciences
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Life sciences study design
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Sample size
The sample size was not predetermined.Diagnostic laboratories were informed about the study through presentations at scientific meetings, publications in national specialist journals, via mailing lists of expert societies and by direct contact.All submitted cases were included in the study.Our dataset represents the largest and most comprehensive dataset on the natural occurrence of BoDV-1 infection in animals and humans that has been assembled so far.
Data exclusions Duplicate sequences originating from the same individual as well as sequences previously identified as laboratory contaminants (see Dürrwald   et al., 2006, 2007; Niller et al., 2020) were excluded from the analysis.GenBank-derived sequences not covering at least the N, X and P genes were likewise not included in the analysis.

Replication
All diagnostic methods were performed following established standard protocols, as described in the materials and methods section, and suitable positive and negative controls were employed to confirm the reliability of each assay.Detection of BoDV-1 RNA was performed by two RT-qPCR assays in parallel.Phylogenetic analyses were performed with the indicated numbers of bootstrap replicates and bootstrap values are provided in the figures.The outlier definition was performed based in parallel based on pairwise nucleotide identities and patrictic distances and both analyses provided consistent results.