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sampled squirrels were from varying locations : private holdings with up to 45 animals per holding , zoological gardens , or roadkill ( table 1 ) .
locations represented 3 countries : germany ( 399 samples from 28 holdings ) , the netherlands ( 49 samples from 4 holdings ) , and the united kingdom ( 20 samples from roadkill ) .
most live squirrels were sampled at the request of the owners . at least 1 swab sample and ,
if possible , duplicates of oral swab samples were collected from living animals , and brain samples were tested from dead squirrels .
blood samples ( edta / heparin ) were also available from 164 squirrels ( table 1 ) .
we obtained congruent results when we tested swabs and brain samples by 2 different vsbv-1specific quantitative reverse transcription pcr ( qrt - pcr ) assays ( 11 ) .
blood samples were centrifuged , and the resulting plasma was tested for the presence of viral rna by using qrt - pcr and for bornavirus - specific igg by using an indirect immunofluorescence assay ( 11 ) . * * swab samples were taken from the oral cavity of each investigated animal . oral swab samples and , if available , brain samples were tested by rt - qpcr .
blood samples ( in edta or heparin ) , wherever available , were tested by quantitative reverse transcription pcr as well as by indirect immunofluorescence assay .
b , blood , bm , brain material ; ss , swab sample ; vsbv-1 , variegated squirrel bornavirus 1 .
we detected vsbv-1 rna and bornavirus - specific antibodies in 11 ( 2.6% ) of the 468 animals .
these 11 vsbv-1positive squirrels ( 7 male , 4 female ) belonged to the family sciuridae , subfamilies sciurinae ( variegated squirrels , s. variegatoides ; 6 animals ) or callosciurinae ( prevost s squirrel , c. prevostii ; 5 animals ) ( table 1 ) .
of the 11 vsbv-1positive animals , 9 were from 4 squirrel breeders in germany ( holdings i  iv ) .
the remaining 2 were from a private holding in the netherlands ( holding v ) .
of the 6 variegated squirrels , 3 originated from holding i , where the first vsbv-1positive animal was identified in 2014 ( 11 ) , and the remaining 3 were from holdings ii and iii .
of the 5 vsbv-1positive 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 20112012 , 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 ) .
histopathology showed that 5 of 9 vsbv-1positive 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-1positive 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 .
immunohistochemical detection of bornavirus x protein ( a ) and phosphoprotein ( b ) in hippocampal neurons of a brain of a prevost s squirrel ( callosciurus prevostii ) collected in germany in 2015 .
inset shows intranuclear dot ( inclusion body ) in cells with and without cytoplasmic immunostaining ( arrows ) .
no staining was observed for bornavirus x protein ( c ) or phosphoprotein ( d ) in a bornavirus - negative variegated squirrel .
original magnification 400 * data are represented in vsbv-1 genome equivalent copies per milliliter of template : + + + , > 10 ; + + , 1010 ; + , 1010 ;  1010 ) . the vsbv-1specific mix 10 quantitative real- time pcr assay was used for quantification ( limit of detection 1 genome equivalent/l template ) .
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 .
accession numbers of the vsbv-1 sequences from this study are lt 594381lt5943919 ( european nucleotide archive ) .
genomes of viruses detected in squirrels from holding i showed the highest similarity with 16 nt substitutions , resulting in 13 aa changes , whereas the other sequences exhibited more variability .
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 ) .
analysis of 11 newly identified vsbv-1 genomes from squirrels collected in germany and the netherlands , 2015 , in comparison with related bornaviruses .
a ) new sequences aligned with published squirrel - derived vsbv-1 genome ( genbank accession no .
g , glycoprotein ; l , large structural protein ; m , matrix ; n , nucleoprotein ; p , phosphoprotein ; x , nonstructural protein .
b ) phylogenetic tree of vsbv-1 isolates from this study ( labeled ) and comparison sequences .
ger , germany ; nl , the netherlands ; vsbv-1 , variegated squirrel bornavirus 1 .
we aligned the 11 vsbv-1 sequences with those from other bornaviruses by using mafft ( multiple alignment using fast fourier transform ) , and we used the best - fit model tim2+i+g4 to construct a phylogenetic tree ( iq - tree version 1.3.10 ; http://iqtree.cibiv.univie.ac.at ) with 1,000 bootstrap replicates . the novel vsbv-1 genomes clustered with the 2 human- and squirrel - derived prototype sequences ln 713680 and ln71368 , forming a unique vsbv-1 clade separate from other mammal , avian , and snake bornaviruses ( sequence identity < 69% ; figure 2 , panel b )
we screened 468 squirrels and identified 11 vsbv-1positive animals , including squirrels belonging to 2 subfamilies of the family sciuridae . although the vsbv-1positive squirrels originated from different holdings and belonged to different subfamilies , the viral genome sequences formed a distinct vsbv-1 cluster .
whole - genome analyses provided no evidence for specific mutation patterns with regard to zoonotic potential or species - specific adaptations .
highest viral genome loads were 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 .
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 .