Molecular epidemiology of human Borna disease virus 1 infection revisited

ABSTRACT Borna disease virus 1 (BoDV-1) strains attracted public interest by recently reported rare fatal encephalitis cases in Germany. Previously, human BoDV-1 infection was suggested to contribute to psychiatric diseases. Clinical outcomes (encephalitis vs. psychiatric disease) and epidemiology (zoonotic vs. human-to-human transmission) are still controversial. Here, phylogenetic analyses of 18 human and 4 laboratory strains revealed close genomic homologies both in distant geographical regions, and different clinical entities. Single unique amino acid mutations substantiated the authenticity of human strains. No matching was found with those of shrew strains in the same cluster 4, arguing against zoonosis. Opposite epidemiology concepts should be equally considered.

Borna disease virus 1 (BoDV-1) strains are key viruses of the species Mammalian 1 orthobornavirus within the ancient family Bornaviridae. Their nonsegmented negative-strand RNA genome (8.9 kb, 6 proteins) replicates in the nucleus of infected cells [1]. BoDV-1 preferentially infects the limbic system of the brain and establishes persistent infections [2].
Human isolates were reported to be recovered from peripheral blood mononuclear cells (PBMCs) [3] and brain [4] of German and Japanese psychiatric patients. Whether a virus may contribute to mental disorders, triggered global research, but remained contraposing, despite global clues [5]. Unusually high genomic homology of BoDV-1 viruses (>95%) [1,6] facilitated doubts about human isolates. They were contradicted by sequence identity between sample and isolate [7], and single amino acid (aa) mutations [3,7] vs. laboratory strain V [1].
Here, we re-evaluated available whole genomes of human strains. Furthermore, we analysed amino acid (aa) translates of human and shrew strains with high nucleotide homology.
Notably, none of the 10 unique shrew mutations (Table S3, supplement) were matching with any of those in human strains. Each strain, either of human or shrew origin, differed by an individual aa-mutation pattern, despite 98% nucleotide homology. For example, encephalitis cases ER2 [10] and P3 [11], both located in Northern Bavaria, shared none of their nine unique aa mutations. Very few mutations (two each in G-and L-pol protein), were previously shown to elicit enhanced neurovirulence in rats [15]. Similarly, Hu-H1 and Hu-H2, isolated from PBMCs of a patient with bipolar depression and obsessivecompulsive disorder, respectively [3], promoted apoptosis and inhibited cell proliferation, contrasting opposite effects by str. V [16,6], despite differing by only few aa-mutations. The Japanese strains, huP2br, isolated from the brain of a schizophrenic patient [4], and Hu-BV differed from each other and H1766 by five unique mutations (Figure 2).

Conclusion
First, human BoDV-1 genome analyses confirmed sequence similarities between distant geographic regions [6] suggesting global prevalence [5] rather than narrow endemic areas [8][9][10][11][12][13]. Second, strains in cluster-4 indirectly suggested a broad clinical spectrum. Third, amino acid analyses demonstrated the Figure 2. Evaluation of amino acid mutations of BoDV-1 strains. Amino acid (aa) changes of seven human and two laboratory cluster-4 viruses vs. strain V using the one-letter-code. Background colour in turquoise indicates all single changes, in lilac non-conservative single changes. *P indicates a mistake in sequence U04608. P 26 S is the correct reading. S26/S28 is the major phosphorylation site of P-protein. For aa alignments, see Table S2. authenticity of human strains by individual mutation signatures. Fourth, no match occurred between unique aa-mutations of shrews and those of human strains, arguing against zoonosis. Fifth, opposite epidemiology concepts should be considered equally, namely zoonotic versus human-to-human transmission driven by unnoticed healthy carriers [5].