Approximately 75% of the emerging pathogens originate from wildlife animals. Retrospective studies performed worldwide on data from 1940 to 2004 have shown a continuing upward trend [1, 2]. Due to the biological relevance of their host species, rodent-borne pathogens beneath bat-associated viruses seem to play the most important role in the emergence of pathogens [5, 32, 33]. This hypothesis seems to be supported by the ongoing reports of newly described rodent-associated agents such as Ljunganvirus or new herpesviruses. Sufficient wild rodent cell culture models are needed in order to enable reliable investigation of these pathogens. Among the rodent species and associated pathogens described in Europe, Myodes glareolus belongs to some of the most relevant ones. In this paper we therefore describe the establishment of a cell line from kidneys of Myodes glareolus.
The appearance of the BVK168 cell line, which was isolated from bank vole kidney by single cell cloning, showed a typical epithelial morphology. The epithelial phenotype was further confirmed by the immunostaining of differentially expressed proteins. Intermediate filament proteins and adhesion molecules serve as markers for specific cell types. Cytokeratins are almost exclusively expressed in epithelia [34, 35]. Loss of cytokeratin and E-cadherin expression and up-regulation of vimentin expression in epithelia correlates with decreased differentiation and epithelial to mesenchymal transition (EMT). However, co-expression of cytokeratin and vimentin was also observed in normal epithelium without signs of dedifferentiation or EMT particularly in epithelia derived from the urogenital tract and in cultured epithelial cell lines . The transformation of epithelial cells to a fibroblastic phenotype is accompanied by the downregulation of tight and adherens junction-proteins . As demonstrated by immunofluorescence, BVK168 cells express the epithelial marker proteins cytokeratin 18 and vimentin. The tight junction-proteins ZO-1, occludin and the adherens junction-proteins E-cadherin and β-catenin are expressed and localized at the cell-cell contacts. In contrast, BVK168 cells did not express proteins specific for endothelial cells or fibroblasts. Therefore, the characterization of the BVK168 cell line reveals a typical epithelial phenotype.
The established BVK168 cell line has so far successfully been propagated to the 49th passage without the need of an immortalization procedure. Spontaneous rodent cell immortalization has been previously reported from many mouse cell lines of different tissue origin and the underlying mechanisms have been investigated for some of these lines [38–42].
The established cell line was highly susceptible to nine very different lab-adapted virus strains of six virus families. For the rhabdovirus VSV and the poxviruses VACV and CPXV it is well known that these viruses have a broad host range in vitro. VSV and CPXV can be grown on a variety of cell lines, e.g. different mammalian or mosquito cells, embryonated chicken eggs, and suckling mice or weanling mice [43–45]. For VACV it was further described that it even propagates in amphibian cells such as frog neurons [46, 47]. Our results for BVK168 cells are therefore in line with these findings.
Following the emergence of USUV in Austria in 2001, a detailed analysis of viral multiplication in 13 permanent cell lines, 3 primary cell cultures, and chicken embryos was published . While chicken embryo fibroblast cells and chicken embryos were resistant to infection, the flavivirus induced a visible cytopathic effect in Vero, PK-15, and goose embryo fibroblast cells. However, in other cell types as hamster (BHK-21, BF), rat (C6) cell lines virus antigen only could be shown by immunohistochemical tests . Given this context, it is remarkable to observe a generalized CPE in BVK168 cells for USUV.
SINV as the alphavirus prototype has been intensively studied for its growth characteristics in a broad range of vertebrate and also invertebrate cells with barely a cell line which did not support its propagation [18, 49]. Susceptibility of cell lines for Inkoo virus was shown for baby hamster kidney, mosquito and renal African green monkey cell lines . Viruses of the California encephalitis serogroup such as Inkoo virus are pathogenic for suckling lab mice and have serologically been detected in bank voles in nature [20, 21]. PIXV was shown to infect mouse embryo cells , spiny rat (Proechimys semispinosus) with low viremia  and to induce pathogenic changes in brain and spleen of white mice [21, 51]. Propagation of PIXV and Inkoo virus in BVK168 cells is therefore in line with results of growth in rodents or rodent cell lines described previously.
Surprisingly, propagation of PUUV strain Vranica in BVK168 failed although this virus strain originally derived from bank voles. This was tested for low and high passages of BVK168 (data not shown in detail) and failed in all performed experiments. The method of choice for isolation of PUUV from rodents currently is its propagation in bank voles in colony [5, 52]. For the strain Kazan it was shown that a Vero E6-adapted strain replicated in these cells to a high efficiency, but did not reproducibly infect bank voles any more. These findings were a consequence of the accumulation of point mutations in S and L gene [53, 54]. This might also be true for the PUUV strain Vranica used in this study. PUUV Vranica is also well adapted to Vero E6 tissue cultures and may have undergone mutations during adaption. Trials to isolate PUUV from PUUV-positive bank voles from South Bavaria  in different passages (8th, 16th, 35th passage) of BVK168 in parallel with Vero E6 cell lines by several sub-passages failed (data not shown). Generally, hantaviruses grow slowly and require up to 10 days to reach 100% infection depending on the adaptation level of the virus strain, infectious doses, and the cell type used. Isolation of hantaviruses proved to be often difficult, requiring several blind passages of the inoculated Vero E6 cell cultures during which the virus adapts to cell culture. The molecular mechanism and genetic basis of the slow growth and adaptation of the virus in cell culture are poorly understood. We revealed no PUUV propagation in the established BVK168 cell line. In comparison, Temonen & coworkers  could show that out of several human cell lines and established bank vole primary cultures exclusively primary bank vole kidney cells could propagate the PUUV virus strain Sotkamo as efficiently as Vero E6 cells with approximately 80% positive cells at day 7 p.i.. However, primarily isolated kidney cells are a pool of different cell types from this tissue. Therefore these may have quite different properties than our BVK168 cell line that has been established by clonal outgrowth of kidney cells. Although meanwhile there exist data on the urine excretion of PUUV in experimentally infected bank voles , to the authors' knowledge the mechanism of propagation of the virus in the voles kidney and the relevant cells have not been investigated in detail. Infections in humans are well known to take place in macrophages and vascular endothelial cells of kidney and lung [57, 58]. As the established BVK168 cells were gained by clonal outgrowth, we cannot exclude that these represent a cell type of vole kidneys that cannot be infected with PUUV. Further experiments e.g. also including antibodies against non-structural proteins or cell compartments have to prove where the block during replication of PUUV in BVK168 is elapsing.
Many epidemiological facts point towards small rodents as BDV reservoir hosts . However, BDV was not yet isolated from naturally infected rodents. Only in one Eulipotyphla (former insectivores) species, the bicolored white-toothed shrew (Crocidura leucodon), BDV could be detected by TaqMan real-time RT-PCR and by immunohistology without evidence of inflammation or degenerative processes in the brain . So far multiple studies have been performed to show the presence of infectious BDV in tissues or blood of human or animal patients. However there are limited methods for the detection of BDV and also only few cell cultures for propagation of BDV [59, 60]. Therefore the new established BVK168 cell lines also could be implemented in future investigations of BDV in neurological human disorders.