Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.     

Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.     

Cultured peripheral neuroglial cells are highly permissive to sheep prion infection

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system (CNS) by prions. Spreading of the infectious agent through the peripheral nervous system (PNS) may represent a crucial step toward CNS neuroinvasion, but the modalities of this process have yet to be clarified. Here we provide further evidence that PNS glial cells are likely targets for infection by prions. Glial cell clones originating from dorsal root ganglia of transgenic mice expressing ovine PrP (tgOv) and simian virus 40 T antigen were found to be readily infectible by sheep scrapie agent. This led us to establish two stable cell lines that exhibited features of Schwann cells. These cells were shown to sustain an efficient and stable replication of sheep prion based on the high level of accumulation of abnormal PrP and infectivity in exposed cultures. We also provide evidence for abnormal PrP deposition in peripheral neuroglial cells from scrapie-infected tgOv mice and sheep. These findings have potential implications in terms of designing new cell systems permissive to prions and of peripheral pathobiology of prion infections.     

Strain‐specific viral properties of variant Creutzfeldt–Jakob disease (vCJD) are encoded by the agent and not by host prion protein

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK‐BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild‐type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain‐specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain‐determining molecules of TSE infectious particles. J. Cell. Biochem. 106: 220–231, 2009. © 2008 Wiley‐Liss, Inc.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Ovine scrapie: priorities and importance

Ovine and caprine scrapie occupies a unique place among animal transmissive spongiform encephalopathies (TSE). It is an object of intensive biomedicinal, ecological and economical studies. Its causative agents are demonstrably associated with the development of TSE in farmed minks, goats and moufflons. Ovine strains of scrapie occurring in North America (particularly in the USA) differ from strains which occur in Europe and were present at the onset of development of TSE in three species of deer living in free nature and in captivity in the USA. The studies dealing with the development of bovine spongiform encephalopathy (BSE) of the English type have indicated justifiably that its origin is associated with one (or more) heretofore unidentified ovine strain. The development of a variant form, the Creutzfeldt-Jacob disease in humans, and transmission of the BSE agent to several families of bovidae, felidae and primates, puts stress on its zoonotic potential. All this leads to the conclusion that domesticated sheep are the decisive reservoir species of animal TSE. They have been infected to an unknown extent with the causative agent of BSE probably through contaminated meat-bone meal. The occurrence of natural ovine prion isolates with properties similar to those of the BSE agent requires that scrapie should be included in the surveillance of human and animal TSE. At present, scrapie is a noticeable disease also in other thanEuropean Communities Member States. It is on the list B of theInternational Epizootics Office. Many countries have initiated control of ovine scrapie. It should therefore become a topical question also in Central and Eastern European countries. Elimination or even eradication of ovine scrapie (or its causative agents) from populations of small and large domestic ruminants is the prerequisite for prevention of penetration of ovine pathogenic prions into the human feed chain. Moreover, it should be ensured that these species will be able to produce foods of a new type (immunotrition and similar) or proteins with therapeutic effects in the near future. Our study established that the PrP genotype of Valachian rams, the Slovak autochthonous breed, contains also VRQ and ARQ alleles encoding the susceptibility to scrapie. Their selection is part of the improvement of Slovak Valachian sheep towards resistance to scrapie.     

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene

We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.     

朊病毒研究的现状

朊病毒疾病是人和动物中的一种传染性,散发性和遗传性的神经退行性脑病,到目前为止,关于此病的致病机制并不十分清楚,但有研究表明该病是由一种正常蛋白PrP的不正常折叠形式在大脑中积聚所致,许多哺乳动物和鸟类的朊病毒基因和氨基酸序列都已被分析,而且传染源的部分性质已经被阐明,朊病毒疾病的传染和潜伏期在人和动物中有很大差异,一些变异与疾病的自发性,易感性和抵抗性有关,为得到关于现毒更多的信息,已对其基因两端进行了大范围的DNA测序分析,至于正常细胞蛋白PrP的生理功能,现在也并不确定。     

Cre-loxP mediated control of PrP to study transmissible spongiform encephalopathy diseases

Expression of the PrP glycoprotein is essential for the development of the transmissible spongiform encephalopathy (TSE) or prion diseases. Although PrP is widely expressed in the mouse, the precise relevance of different PrP-expressing cell types to disease remains unclear. To address this, we generated two lines of floxed PrP gene-targeted transgenic mice using the Cre recombinase-loxP system. These floxed mice allow a functional PrP allele to be either switched "on" or "off." We demonstrate control of PrP expression for both alleles following Cre-mediated recombination, as determined by PrP mRNA and protein expression in the brain. Moreover, we show that Cre-mediated alteration of PrP expression in these mice has a major influence on the development of TSE disease. These floxed PrP mice will allow the involvement of PrP expression in specific cell types following TSE infection to be defined, which may identify potential sites for therapeutic intervention.     

Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes

AIMS: To analyse the frequencies of prion (PrP) gene haplotypes in UK sheep flocks and evaluate their relevance to transmissible spongiform encephalopathies (TSEs) and TSE resistance breeding programmes in sheep. METHODS AND RESULTS: Genomic DNA isolated from sheep blood was PCR amplified for the coding region of the PrP gene and then sequenced. This study has analysed the sequence of PrP between codons 110 and 245 in 6287 ARQ haplotypes revealing a total of eight variant sequences, which represent a higher than expected 41% of all ARQ haplotypes. The additional PrP gene dimorphisms were M112T, L141F, M137T, H143R, H151C, P168L, Q175E and P241S. CONCLUSION: The results do not suggest a correlation between the occurrence of a specific ARQ haplotype and the scrapie disease status of a flock. The ARQ haplotype variability appears to be different in the UK sheep flocks compared with sheep flocks from outside the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional PrP dimorphisms may impact on the methodologies used for standard PrP genotyping in sheep breeding programmes. Some of these polymorphisms were found with significant frequencies in the UK sheep flocks and should therefore be considered in breeding programmes.     

PrP polymorphisms tightly control sheep prion replication in cultured cells

Prion diseases are fatal neurodegenerative disorders of animals and humans that are characterized by the conversion of the host-encoded prion protein (PrP) to an abnormal isoform. In several species, including humans, polymorphisms in the gene encoding the PrP protein tightly control susceptibility of individuals toward this disease. In the present study we show that Rov cells expressing an ovine PrP allele ((VRQ)PrP) associated with high susceptibility of sheep to scrapie were very sensitive to sheep prion transmission and replicated the agent to high titers. In contrast, we did not find any evidence of infection when Rov cells expressed similar levels of a PrP variant ((ARR)PrP) linked to resistance. Our data provide the first direct evidence that natural PrP polymorphisms may affect prion susceptibility by controlling prion replication at the cell level. The study of how PrP polymorphisms influence the genetic control of prion propagation in cultured Rov cells may help elucidate basic mechanisms of prion replication.     

Atypical/Nor98 scrapie infectivity in sheep peripheral tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.     

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.     

Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).     

Infectious prions and proteinopathies

Transmissible spongiform encephalopathies (TSEs) are caused by an infectious agent that is thought to consist of only misfolded and aggregated prion protein (PrP). Unlike conventional micro-organisms, the agent spreads and propagates by binding to and converting normal host PrP into the abnormal conformer, increasing the infectious titre. Synthetic prions, composed of refolded fibrillar forms of recombinant PrP (rec-PrP) have been generated to address whether PrP aggregates alone are indeed infectious prions. In several reports, the development of TSE disease has been described following inoculation and passage of rec-PrP fibrils in transgenic mice and hamsters. However in studies described here we show that inoculation of rec-PrP fibrils does not always cause clinical TSE disease or increased infectious titre, but can seed the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). These data are reminiscent of the “prion-like” spread of misfolded protein in other models of neurodegenerative disease following inoculation of transgenic mice with pre-formed amyloid seeds. Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity.     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.     

Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells.

The immune system is central in the pathogenesis of scrapie and other transmissible spongiform encephalopathies (TSEs) or 'prion' diseases. After infecting by peripheral (intraperitoneal or oral) routes, most TSE agents replicate in spleen and lymph nodes before neuroinvasion. Characterization of the cells supporting replication in these tissues is essential to understanding early pathogenesis and may indicate potential targets for therapy, for example, in 'new variant' Creutzfeldt-Jakob disease. The host 'prion' protein (PrP) is required for TSE agent replication and accumulates in modified forms in infected tissues. Abnormal PrP is detected readily on follicular dendritic cells (FDCs) in lymphoid tissues of patients with 'new variant' Creutzfeldt-Jakob disease, sheep with natural scrapie and mice experimentally infected with scrapie. The normal protein is present on FDCs in uninfected mice and, at lower levels, on lymphocytes. Studies using severe combined immunodeficiency (SCID) mice, with and without bone marrow (BM) grafts, have indicated involvement of FDCs and/or lymphocytes in scrapie pathogenesis. To clarify the separate roles of FDCs and lymphocytes, we produced chimeric mice with a mismatch in PrP status between FDCs and other cells of the immune system, by grafting bone marrow from PrP-deficient knockout mice into PrP-expressing mice and vice versa. Using these chimeric models, we obtained strong evidence that FDCs themselves produce PrP and that replication of a mouse-passaged scrapie strain in spleen depends on PrP-expressing FDCs rather than on lymphocytes or other bone marrow-derived cells.