Variegated Squirrel Bornavirus 1 in Squirrels, Germany and the Netherlands

We screened squirrels in Germany and the Netherlands for the novel zoonotic variegated squirrel bornavirus 1 (VSBV-1). The detection of VSBV-1 in 11 squirrels indicates a considerable risk for transmission to humans handling those animals. Therefore, squirrels in contact with humans should routinely be tested for VSBV-1.

animal was identified in 2014 (11), and the remaining 3 were from holdings II and III. Squirrels from the 3 holdings were traded for breeding. Of the 5 VSBV-1-positive Prevost's squirrels, 3 came from holding IV in Germany, and 2 were from holding V. The positive squirrels in the Netherlands had been transferred from a breeder in Germany in 2011-2012, but we could establish no direct epidemiologic link to the holdings in Germany where we found VSBV-1positive animals.
All animals with detectable virus as well as 40 negative squirrels from the same holdings were euthanized and checked for macroscopic lesions. We applied hematoxylin and eosin staining to formalin-fixed, paraffin-embedded brain sections from 9 of 11 animals and performed immunohistochemistry as described previously (12). A BoDVpositive horse brain sample served as positive control. Histopathology showed that 5 of 9 VSBV-1-positive animals, variegated squirrels as well as Prevost's squirrels, had  mild nonsuppurative meningitis or encephalitis. In 8 of 9 animals, we also detected intranuclear eosinophilic Joest-Degen inclusion bodies in scattered neurons of brain, spinal cord, and trigeminal as well as spinal ganglia (data not shown). We observed bornavirus-specific phosphoprotein and X protein throughout the brain in neurons, glial cells, and in a few ependymal cells in nuclei, cytoplasm, and cellular processes ( Figure 1). We tested a panel of organ samples from all euthanized animals by qRT-PCR. All animals for which swab samples were VSBV-1-positive harbored considerable VSBV-1 genome loads, whereas animals for which swab samples were negative for viral RNA were also negative for viral loads in all tested organs (Table 2; data for control animals not shown). We found the highest viral RNA loads in the CNS and organs (kidney, nose, bladder, salivary gland, and sex organs), which could play a role in viral shedding and transmission ( Table 2). Skin sections were also positive, indicating that VSBV-1 has broad cell and organ tropism. We were able to cocultivate infected primary squirrel cells with a permanent cell line and to isolate infectious virus from these passaged cells.
We sequenced the VSBV-1 genome (8,786 nt; missing only the 5′ and 3′ noncoding regions) from all 11 squirrels and compared the sequences with the published VSBV-1 prototype sequence from a variegated squirrel (GenBank accession no. LN713680) (11). Accession numbers of the VSBV-1 sequences from this study are LT 594381-LT5943919 (European Nucleotide Archive).
Genomes of viruses detected in squirrels from holding I showed the highest similarity with 1-6 nt substitutions, resulting in 1-3 aa changes, whereas the other sequences exhibited more variability. We found <29 nt substitutions causing <19 aa changes. The mutations were evenly distributed over the coding regions, but in viral genomes of squirrels from the same holding, they often occurred at the same position (Figure 2, panel A).

Conclusions
We screened 468 squirrels and identified 11 VSBV-1-positive animals, including squirrels belonging to 2 subfamilies Table 2. VSBV-1 RNA levels in samples from squirrels collected in Germany and the Netherlands in 2015 that were positive for viral RNA and VSBV-1 antibodies*

Animal and sample type
Country and holding no. Germany The Netherlands  I  I  I  II  II  III  IV  IV  IV  V    found in the CNS, followed by the oral cavity and skin, indicating the potential for transmission to humans through scratching or biting. Only those squirrels positive for VSBV-1 by qRT-PCR displayed bornavirus-specific antibodies. Although serologic analyses support the qRT-PCR results, collecting serum samples is difficult and often not feasible for private breeders. Our data suggest that screening of swab samples is a suitable and reliable tool for noninvasive monitoring of squirrels for VSBV-1 infection. The prevalence of 3.3% for S. variegatoides and 8.8% for C. prevostii squirrels indicates a considerable risk for transmission to humans handling those animals without taking precautionary measures. We therefore recommend routine testing of squirrels in contact with humans, such as those in breeding and holding facilities or zoological gardens, at least from the subfamilies Sciurinae and Callosciurinae.