Introduction
Prion diseases (transmissible spongiform encephalopathies [TSEs]) are progressive, fatal, transmissible, neurodegenerative diseases, which include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and various forms of Creutzfeldt-Jakob disease (CJD) and kuru in humans [1]. The similarities between scrapie and CJD have long been recognized [2], and scrapie is the prototypical prion disease [3]; thus, scrapie is an experimental model that allows for the investigation of a natural prion disease in a natural host. The central feature of prion pathogenesis is the conversion of the normal cellular form of the host-encoded prion protein (PrPC [C superscript for cellular]) to an abnormal isoform, designated PrPSc (Sc superscript for sheep scrapie) [4,5,6]. This post-translational conversion involves a conformational change resulting in a detergent-insoluble, partially protease-resistant molecule that aggregates in affected cells and serves as the marker for prion diseases. PrPSc-accumulating cellsinclude neurons and monocyte-derived cells (macrophages, microglia, and dendritic cells), among others [7,8,9,10,11]. Studies to identify anti-prion compounds often initially rely on inhibition of in vitro PrPSc formation [12]. Currently, however, there are no effective treatments for prion diseases despite abundant investigation into therapeutics [43,44,45]. Continued investigation into new classes of anti-prion compounds is thus warranted, not only for the development of effective in vivoanti-prion molecules, but also as research tools to elucidate the cellular pathogenesis of prion diseases. Most of the studies to detect anti-prion compounds have used rodent cell culture systems with rodent-adapted prion strains. While these rodent models have many benefits, attempts have been made at improving upon them.

Rov9 cells are rabbit renal epithelial cells (RK-13) that have the 136VV/154RR/171QQ allele of the sheep PRNP gene under control of a doxycyclineinducible promoter and accumulate sheep-derived prions [46]. Using these more natural, yet still far from completely natural, cells it has been shown that anti-prion compounds identified using rodent-adapted PrPSc systems often fail to demonstrate anti-prion activity when using sheep-origin PrPSc [47]. The inability of these compounds to specifically inhibit sheep-derived prions suggests the importance for even more natural prion models for anti-prion compound screening as the species of origin or cell type may also impact the results. Currently there are only two cell culture models that are derived from a natural TSE host, a mule deer-derived brain fibroblast cell line susceptible to PrPCWD [48] and a sheepderived microglial cell system susceptible to sheep-origin PrPSc [49]. The mule deer-derived brain fibroblast cell line has been used to demonstrate the anti-prion activity of pentosan polysulfate and a porphyrin compound [48]. Besides the varying effectiveness of anti-prion compounds in different systems, another consideration for model development is the potential for significant effects of co-infecting agents. It has been shown that small ruminant lentivirus infection is associated with enhanced distribution of PrPSc in naturally co-infected sheep [50,51]. This effect in sheep may be related to virus-enhanced, intracellular accumulation of PrPSc, as has been demonstrated in vitro using primary sheep microglial cells [49]. It is unknown if other virus families have similar effects. Flaviviruses are a group of enveloped, positive-sense stranded RNA viruses that can infect monocyte-lineage cells, establish persistent infections in vivo, and establish noncytopathic infections in vitro [52,53,54]. Rov9 cells, as derivatives of RK13 cells [55], and sheep microglial cells (data reported herein) are susceptible to bovine viral diarrhea virus (BVDV, genus Pestivirus, family Flaviviridae ) infection. To cure cells of a potentially confounding, co-infecting virus, 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772; Fig. 1), a known BVDV inhibitor [56,57], was used. In addition to inhibiting BVDV, this treatment also inhibited PrPSc accumulation. Here we describe the anti-prion activity of DB772, a monocationic phenyl-furanbenzimidazole [58], which belongs to a chemical category previously untested for anti-PrPSc activity. In summary, only one study has investigated in vitro chemical inhibition of prions in a cell system derived from a natural host [48] and no studies have tested for anti-prion activity in a sheep cell culture system or in microglial cells from any species, despite the relevance of sheep scrapie and monocyte-derived cells (e.g., microglia) to prion diseases. Reported herein is the discovery ofanti-prion activity of a compound belonging to a previously untested chemical category using sheep-origin PrPSc and sheep microglial cells.

Materials and Methods Ethics Statement
The Institutional Animal Care and Use Committee at Washington State University approved this study protocol (Permit numbers: #03811 and 03987). The ewe was euthanized by administering an intravenous overdose of sodium pentobarbital, in accordance with the 2007 American Veterinary Medical Association Guidelines on Euthanasia, and all efforts were made to minimize suffering.

Cells
Primary sheep microglial cells were obtained from a near-term Suffolk-cross fetus and cultured as previously described [49]. All cell media were made with pestivirus-free, fetal-bovine serum. Microglial cells were phenotyped via immunocytochemistry using the microglial markers biotinylated Ricinus communis agglutinin-1 (RCA-1) (Dako Cytomation) and an anti-CD14 antibody (MM61A, IgG1, VMRD, Inc.), as previously described [49]. A pellet of microglial cells was collected, washed by centrifugation, and used for genotyping the fetal prion gene as previously described [59]. Rov9 cells (B. Caughey with permission from D. Vilette) are rabbit renal epithelial cells (RK-13) stably transfected with the sheep VRQ (Val-136, Arg-154, Gln-171) allele of the prion gene under the control of a tetracycline-inducible promoter [46]. Rov9 cells were maintained in OMEM supplemented with 1 mg/ml doxycycline (OMEM-Doxy), as previously described [46]. PrPSc within Rov9Sc cells was verified by PrPSc-specific enzyme-linked immunosorbent assay (ELISA) (see below). Since Rov9 cells are derived from RK13 cells, Rov9 cells are permissive to BVDV infection [55]. Prior to inoculation microglial cells were confirmed BVDV negative and Rov9 cells were confirmed BVDV positive by RT-PCR and BVDV antigen ELISA (see below). The scrapie inoculum also contains infectious BVDV, and the preparation and application of PrPSc inoculum also transmits BVDV. Untreated microglial cells were used as controls for BVDV contamination.