Infections of horses and shrews with Bornaviruses in Upper Austria: a novel endemic area of Borna disease

Abstract

Borna disease, a lethal infection with Borna disease virus-1 (BoDV-1), was diagnosed in four horses from Upper Austria in 2015 and 2016. All cases occurred in winter (two cases in February 2015 and two cases in December 2016), and the maximal distance of the affected stables was 17 km. To demonstrate whether the causative agent was also harbored by its reservoir host, the bicolored white-toothed shrew (Crocidura leucodon), 28 shrews from this geographic area were collected in 2015 and investigated for the presence of BoDV-1. The shrew species were identified according to taxonomic clues and molecular barcodes. Affected horses and all shrews were investigated using histology, immunohistochemistry (IHC) and reverse transcription PCR. The horses exhibited severe nonpurulent encephalitis. Large amounts of BoDV-1 antigen were identified in their CNS. Among the 28 shrews, nine were identified as C. leucodon and 13 as Sorex araneus (Common shrew; Eurasian shrew). Six C. leucodon (66.7%) and one S. araneus (7.7%) had BoDV-1 infections. In accordance with previous findings, the IHC of C. leucodon exhibited a high amount of viral antigen in many neural and extraneural tissues. By contrast, the single positive S. araneus had an exclusively neural staining pattern. Of all positive samples, whole-genome BoDV-1 sequences were generated. The acquired sequences of the affected shrews were not identical to each other and clustered around the sequences of the diseased horses belonging, surprisingly, to the German 'strain V' cluster.

Figure 1

Map depicting the geographical distribution of the BoDV-1-positive horses and shrews. The inset shows the area in Upper Austria, which has been enlarged.

Figure 2

Bornavirus encephalitis in horses. (A) Severe nonpurulent encephalitis with perivascular cuffing (asterisk) and microgliosis. Bar=250 μm. (B and C) Eosinophilic intranuclear inclusion bodies (arrows) in a brainstem neuron (B) and a trigeminal ganglion neuron surrounded by lymphohistiocytic infiltration (C). Bars=25 μm. (D and E) Immunohistochemical demonstration of BoDV antigen (brown signals) in neurons and glial cells and their processes. Bars=100 μm. (A–C) Hematoxylin and Eosin staining. (D and E) Bo-18 immunohistochemistry.

Figure 3

Bornavirus infection of shrews. (A and B) Demonstration of BoDV-1 antigen in an infected Crocidura leucodon shrew. In the brain (A), the antigen is diffusely distributed; in skin (B), keratinocytes and periadnexal nerve fibers are positive. (C and D) Demonstration of BoDV-1 antigen in an infected Sorex araneus shrew. In the brain (C), the viral antigen has a multifocal, patchy staining pattern, representing individually stained neurons and glial cells and their processes. In skin (D), the viral antigen is confined to periadnexal nerve fibers; keratinocytes are negative. (E and F) Lack of immunoreactivity in brain (E) and skin (F) in a non-infected C. leucodon shrew. Bars=40 μm; Bo-18 immunohistochemistry.

Figure 4

Phylogenetic tree of the complete coding sequences of representative members of the genus Bornavirus including new Upper Austrian sequences. (A) For better visualization, the eleven sequences determined in this study are collapsed. (B) Only the sequences of the new Upper Austrian Bornaviruses are presented. These sequences are marked with green triangles (horse-derived BoDVs-1), red diamonds (C. leucodon-derived BoDVs-1), and blue diamond (S. araneus-derived BoDVs-1). GenBank accession numbers, strain names (in the case of BoDVs) and species abbreviations are indicated at the branches. All supporting bootstrap values are displayed next to the nodes. The horizontal scale bar indicates genetic distances. *GenBank acc. nos: KY002971-79 and KY490040-41.

Figure 5

Phylogenetic tree of an 1824-bp long sequence stretch (coding for N protein, intergenic N/X, as well as for X and P proteins) of 43 selected BoDVs-1 from the GenBank and eleven sequences determined in this study (which are collapsed). The five major clusters are indicated. The GenBank accession numbers, host or strain names, geographic location and years of isolations are indicated at the branches. Supporting bootstrap values ≥70% are displayed next to the nodes. The horizontal scale bar indicates genetic distances (here 0.5% nucleotide sequence divergence). BA, Bavaria; BW, Baden-Wurttemberg; GB, Graubuenden; HE, Hesse; L, Liechtenstein; RP, Rhineland-Palatinate; SA, Saxony-Anhalt; SG, Sankt Gallen; SX, Saxony; TH, Thuringia. *GenBank acc. nos: KY002971-79 and KY490040-41.