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1.  Immunohistochemical study of PrPSc distribution in neural and extraneural tissues of two cats with feline spongiform encephalopathy 
Background
Two domestic shorthair cats presenting with progressive hind-limb ataxia and increased aggressiveness were necropsied and a post mortem diagnosis of Feline Spongiform Encephalopathy (FSE) was made. A wide spectrum of tissue samples was collected and evaluated histologically and immunohistologically for the presence of PrPSc.
Results
Histopathological examination revealed a diffuse vacuolation of the grey matter neuropil with the following areas being most severely affected: corpus geniculatum medialis, thalamus, gyrus dentatus of the hippocampus, corpus striatum, and deep layers of the cerebral and cerebellar cortex as well as in the brain stem. In addition, a diffuse glial reaction involving astrocytes and microglia and intraneuronal vacuolation in a few neurons in the brain stem was present.
Heavy PrPSc immunostaining was detected in brain, retina, optic nerve, pars nervosa of the pituitary gland, trigeminal ganglia and small amounts in the myenteric plexus of the small intestine (duodenum, jejunum) and slightly in the medulla of the adrenal gland.
Conclusion
The PrPSc distribution within the brain was consistent with that described in other FSE-affected cats. The pattern of abnormal PrP in the retina corresponded to that found in a captive cheetah with FSE, in sheep with scrapie and was similar to nvCJD in humans.
doi:10.1186/1746-6148-5-11
PMCID: PMC2673214  PMID: 19335885
2.  Possible Case of Maternal Transmission of Feline Spongiform Encephalopathy in a Captive Cheetah 
PLoS ONE  2009;4(9):e6929.
Feline spongiform encephalopathy (FSE) is considered to be related to bovine spongiform encephalopathy (BSE) and has been reported in domestic cats as well as in captive wild cats including cheetahs, first in the United Kingdom (UK) and then in other European countries. In France, several cases were described in cheetahs either imported from UK or born in France. Here we report details of two other FSE cases in captive cheetah including a 2nd case of FSE in a cheetah born in France, most likely due to maternal transmission. Complete prion protein immunohistochemical study on both brains and peripheral organs showed the close likeness between the two cases. In addition, transmission studies to the TgOvPrP4 mouse line were also performed, for comparison with the transmission of cattle BSE. The TgOvPrP4 mouse brains infected with cattle BSE and cheetah FSE revealed similar vacuolar lesion profiles, PrPd brain mapping with occurrence of typical florid plaques. Collectively, these data indicate that they harbor the same strain of agent as the cattle BSE agent. This new observation may have some impact on our knowledge of vertical transmission of BSE agent-linked TSEs such as in housecat FSE, or vCJD.
doi:10.1371/journal.pone.0006929
PMCID: PMC2732902  PMID: 19738899
3.  Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States 
PLoS ONE  2010;5(1):e8765.
Background
Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.
Methodology/Principal Findings
Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrPres) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrPres detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrPres found in the brain had the typical characteristics of the PrPres present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrPres was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.
Conclusions/Significance
Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.
doi:10.1371/journal.pone.0008765
PMCID: PMC2808239  PMID: 20098730
4.  Beyond PrPres Type 1/Type 2 Dichotomy in Creutzfeldt-Jakob Disease 
PLoS Pathogens  2008;4(3):e1000029.
Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres) identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability.
Author Summary
Prion diseases are transmissible neurodegenerative disorders characterized by accumulation of an abnormal isoform (PrPSc) of a host-encoded protein (PrPC) in affected tissues. According to the prion hypothesis, PrPSc alone constitutes the infectious agent. Sporadic Creutzfeldt-Jakob disease (sCJD) is the commonest human prion disease. Although considered as a spontaneous disorder, the clinicopathological phenotype of sCJD is variable and substantially influenced by the methionine/valine polymorphism at codon 129 of the prion protein gene (PRNP). Based on these clinicopathological and genetic criteria, a subclassification of sCJD has been proposed. Here, we used two new biochemical assays that identified four distinct biochemical PrPSc subgroups in a cohort of 41 sCJD cases. These subgroups correlate with the current sCJD subclassification and could therefore represent distinct prion strains. Iatrogenic CJD (iCJD) occurs following presumed accidental human-to-human sCJD transmission. Our biochemical investigations on 12 iCJD cases from different countries found the same four subgroups as in sCJD. However, in contrast to the sCJD cases, no particular correlation between the PRNP codon 129 polymorphism and biochemical PrPSc phenotype could be established in iCJD cases. This study provides an alternative biochemical definition of PrPSc diversity in human prion diseases and new insights into the perception of agent variability.
doi:10.1371/journal.ppat.1000029
PMCID: PMC2279301  PMID: 18389084
5.  Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability 
PLoS Pathogens  2014;10(6):e1004158.
The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.
Author Summary
Prions are infectious protein particles causing fatal diseases in mammals. Prions correspond to self-perpetuating amyloid protein polymers. Prions also exist in fungi where they behave as cytoplasmic infectious elements. The [Het-s] prion of the fungus Podospora anserina constitutes a favorable model for the analysis of the structural basis of prion propagation because a high resolution structure of the prion form of [Het-s] is available, a situation so far unique to this prion model. We have analyzed the relation between [Het-s] structure and function using alanine scanning mutagenesis. We have generated 32 single amino acid variants of the prion forming domain and analyzed their prion function in vivo and structure by solid-state NMR. We find that the PFD structure is very robust and that only a few key mutations affect prion structure and function. In addition, we find that a C-terminal semi-flexible loop plays a critical role in prion propagation although it is not part of rigid amyloid core. This study offers insights on the structural basis of prion propagation and illustrates that accessory regions outside of the amyloid core can critically participate in prion function, an observation that could be relevant to other amyloid models.
doi:10.1371/journal.ppat.1004158
PMCID: PMC4055769  PMID: 24945274
6.  Prions in Milk from Ewes Incubating Natural Scrapie 
PLoS Pathogens  2008;4(12):e1000238.
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.
Author Summary
A decade ago, a new variant form of Creutzfeldt-Jakob disease was identified. The emergence of this prion disease in humans was the consequence of the zoonotic transmission of bovine spongiform encephalopathy through dietary exposure. Since then, the control of human exposure to prions has become a priority, and a policy based on the exclusion of known infectious materials from the food chain has been implemented. Because all investigations carried out failed to reveal evidence of infectivity in milk from affected ruminants, this product has continuously been considered as safe. In this study, we demonstrate the presence of prions in colostrum and milk from sheep incubating natural scrapie and displaying apparently healthy mammary glands. This finding indicates that milk from small ruminants could contribute to the transmission of prion disease between animals. It also raises some concern with regard to the risk to humans associated with milk products from ovine and other dairy species.
doi:10.1371/journal.ppat.1000238
PMCID: PMC2587713  PMID: 19079578
7.  A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice 
PLoS Pathogens  2008;4(8):e1000137.
The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.
Author Summary
The origin of the transmissible agent involved in the food-borne epidemic of bovine spongiform encephalopathy (BSE) remains a mystery. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic, form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. In this study we analyzed the molecular features of the disease-associated protease-resistant prion protein (PrPres) found in the brain of transgenic mice overexpressing the ovine prion protein after experimental infection with prions from bovine classical and L-type BSEs or from ovine scrapie. Scrapie cases included rare “CH1641-like” isolates, which share some PrPres molecular features with classical BSE and L-type BSE. Scrapie isolates induced in transgenic mouse brains the production of a C-terminally cleaved form of PrPres, which was particularly abundant from “CH1641-like” cases. In contrast, this C-terminal prion protein product was undetectable in ovine transgenic mice infected with bovine prions from both classical and L-type BSE. These findings add a novel approach for the discrimination of prions that may help to understand their possible changes during cross-species transmissions.
doi:10.1371/journal.ppat.1000137
PMCID: PMC2516186  PMID: 18769714
8.  H-Type Bovine Spongiform Encephalopathy 
Prion  2007;1(1):61-68.
We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrPres) in Western blot, with a 1–2 kDa higher apparent molecular mass of the unglycosylated PrPres associated with labelling by antibodies against the 86–107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrPres, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrPres (PrPres #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrPres fragment of ≈7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrPres #2 and ≈7 kDa PrPres were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.
PMCID: PMC2633710  PMID: 19164888
prion; BSE; Creutzfeldt-Jakob; Gerstmann-Sträussler-Scheinker; Western blot; amyloid
9.  Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle? 
Background
The epidemic form of Bovine Spongiform Encephalopathy (BSE) is generally considered to have been caused by a single prion strain but at least two strain variants of cattle prion disorders have recently been recognized. An additional neurodegenerative condition, idiopathic brainstem neuronal chromatolysis and hippocampal sclerosis (IBNC), a rare neurological disease of adult cattle, was also recognised in a sub-set of cattle submitted under the BSE Orders in which lesions of BSE were absent. Between the years of 1988 and 1991 IBNC occurred in Scotland with an incidence of 7 cases per 100,000 beef suckler cows over the age of 6 years.
Results
When the brains of 15 IBNC cases were each tested by immunohistochemistry, all showed abnormal labelling for prion protein (PrP). Immunohistological labelling for PrP was also present in the retina of a single case available for examination. The pattern of PrP labelling in brain is distinct from that seen in other ruminant prion diseases and is absent from brains with other inflammatory conditions and from normal control brains. Brains of IBNC cattle do not reveal abnormal PrP isoforms when tested by the commercial BioRad or Idexx test kits and do not reveal PrPres when tested by Western blotting using stringent proteinase digestion methods. However, some weakly protease resistant isoforms of PrP may be detected when tissues are examined using mild proteinase digestion techniques.
Conclusion
The study shows that a distinctive neurological disorder of cattle, which has some clinical similarities to BSE, is associated with abnormal PrP labelling in brain but the pathology and biochemistry of IBNC are distinct from BSE. The study is important either because it raises the possibility of a significant increase in the scope of prion disease or because it demonstrates that widespread and consistent PrP alterations may not be confined to prion diseases. Further studies, including transmission experiments, are needed to establish whether IBNC is a condition in which prion protein is abnormally regulated or it is yet a further example of an infectious cattle prion disease.
doi:10.1186/1746-6148-4-38
PMCID: PMC2569918  PMID: 18826563
10.  Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region 
PLoS ONE  2012;7(12):e50623.
Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrPC) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrPC protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.
doi:10.1371/journal.pone.0050623
PMCID: PMC3517517  PMID: 23236380
11.  Genesis of Mammalian Prions: From Non-infectious Amyloid Fibrils to a Transmissible Prion Disease 
PLoS Pathogens  2011;7(12):e1002419.
The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrPSc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc template. Here we report that authentic PrPSc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrPSc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrPSc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrPSc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.
Author Summary
The transmissible agent of prion disease consists of a prion protein in its abnormal conformation (PrPSc), which replicates itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc. The current study reports that infectious prions and transmissible prion disease can be triggered in wild type animals by amyloid fibrils produced from recombinant prion prtotein, which are structurally different from PrPSc and lacks any detectable PrPSc particles. This work introduces a new hypothesis that transmissible prion diseases can be induced by prion protein structures different from that of authentic PrPSc and suggests that a new mechanism for triggering PrPSc formation different from the classical templating exists. The current work provides important new insight into the mechanisms underlying genesis and evolution of the transmissible states of the prion protein and has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.
doi:10.1371/journal.ppat.1002419
PMCID: PMC3228811  PMID: 22144901
12.  Susceptibility of Domestic Cats to Chronic Wasting Disease 
Journal of Virology  2013;87(4):1947-1956.
Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postinoculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal disease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., and intraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculated cats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (FelCWD) was demonstrated in the brains of all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealed abnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyperintensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of 4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.
doi:10.1128/JVI.02592-12
PMCID: PMC3571486  PMID: 23236066
13.  Highly Efficient Protein Misfolding Cyclic Amplification 
PLoS Pathogens  2011;7(2):e1001277.
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro.
Author Summary
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro. While PMCA has become an important tool in prion research, its application is limited because of low yield, poor efficiency and, sometimes, stochastic behavior. The current study introduces a new PMCA format that dramatically improves the efficiency, yield, and robustness of prion conversion in vitro and reduces the time of the reaction. These improvements have numerous implications. The method opens new opportunities for improving prion detection and for generating large amounts of PrPSc in vitro. Furthermore, the results demonstrate that in vitro conversion is not limited by lack of convertible PrPC nor by concentrations of cellular cofactors required for prion conversion.
doi:10.1371/journal.ppat.1001277
PMCID: PMC3037363  PMID: 21347353
14.  Prions of Ruminants Show Distinct Splenotropisms in an Ovine Transgenic Mouse Model 
PLoS ONE  2010;5(4):e10310.
Background
Transmissible agents involved in prion diseases differ in their capacities to target different regions of the central nervous system and lymphoid tissues, which are also host-dependent.
Methodology/Principal Findings
Protease-resistant prion protein (PrPres) was analysed by Western blot in the spleen of transgenic mice (TgOvPrP4) that express the ovine prion protein under the control of the neuron-specific enolase promoter, after infection by intra-cerebral route with a variety of transmissible spongiform encephalopathies (TSEs) from cattle and small ruminants. Splenic PrPres was consistently detected in classical BSE and in most natural scrapie sources, the electrophoretic pattern showing similar features to that of cerebral PrPres. However splenic PrPres was not detected in L-type BSE and TME-in-cattle, or in the CH1641 experimental scrapie isolate, indicating that some TSE strains showed reduced splenotropism in the ovine transgenic mice. In contrast with CH1641, PrPres was also consistently detected in the spleen of mice infected with six natural “CH1641-like” scrapie isolates, but then showed clearly different molecular features from those identified in the brains (unglycosylated PrPres at ∼18 kDa with removal of the 12B2 epitope) of ovine transgenic mice or of sheep. These features included different cleavage of the main PrPres cleavage product (unglycosylated PrPres at ∼19 kDa with preservation of the 12B2 epitope) and absence of the additional C-terminally cleaved PrPres product (unglycosylated form at ∼14 kDa) that was detected in the brain.
Conclusion/Significance
Studies in a transgenic mouse model expressing the sheep prion protein revealed different capacities of ruminant prions to propagate in the spleen. They showed unexpected features in “CH1641-like” ovine scrapie suggesting that such isolates contain mixed conformers with distinct capacities to propagate in the brain or lymphoid tissues of these mice.
doi:10.1371/journal.pone.0010310
PMCID: PMC2859945  PMID: 20436680
15.  Isolation from Cattle of a Prion Strain Distinct from That Causing Bovine Spongiform Encephalopathy 
PLoS Pathogens  2006;2(10):e112.
To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrPres) isolated from brains of affected animals or humans. However, alternate PrPres signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrPres of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.
Synopsis
Prions are unconventional agents of proteic nature that are formed of abnormal conformations of the host-encoded prion protein (PrP). They cause fatal neurodegenerative diseases in both animals and humans, and can be transmitted between species as exemplified in humans by the emergence of variant Creutzfeldt-Jakob disease following the epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom. Since diagnosis of prion infection is only possible once the central nervous system has been invaded, brains of slaughtered or fallen cattle are routinely screened in Europe to protect the consumers from BSE. This has unexpectedly led to the discovery of unprecedented PrP conformations that were distinct from the single one associated so far with BSE or BSE-related diseases. To precisely determine their etiology, the authors have studied the transmissibility of these new conformations, termed H-type, to transgenic mice expressing either bovine or ovine PrP. They show that these cases are highly pathogenic for these mice. The authors also demonstrate that they are not directly related to the agent involved in the BSE epidemic, supporting the view for isolation of a new prion strain from cattle, whose prevalence and associated zoonotic risk should be carefully monitored in the future.
doi:10.1371/journal.ppat.0020112
PMCID: PMC1617128  PMID: 17054396
16.  Prion Protein Amino Acid Determinants of Differential Susceptibility and Molecular Feature of Prion Strains in Mice and Voles 
PLoS Pathogens  2008;4(7):e1000113.
The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases.
Author Summary
Prions are unconventional infectious agents that cause fatal neurodegenerative diseases in animals and humans. A pathological form of the cellular prion protein (PrPC), named PrPSc, appears to be the major or the sole component of prions. These agents are transmitted by inducing the conversion of host PrPC into PrPSc that accumulates in the brain of affected individuals. Different factors are believed to modulate such events, which explains the variable transmission efficiency observed under inter-species experimental inoculation. These factors are still fairly unknown, although evidence exists that some kind of structural compatibility between PrPSc of the infectious inoculum and PrPC of the host has a role in making transmission more or less efficient. We investigated the transmission of prions to different rodents and showed that specific amino acid substitutions (Y154N and S169N) in the prion protein are major determinants of susceptibility to prions. In particular, we showed that these specific variations i) direct the transmission rate of prions between different species in a way that is dependent on the prion strain, ii) affect the molecular characteristics of prions, and iii) influence their replication efficiency.
doi:10.1371/journal.ppat.1000113
PMCID: PMC2453331  PMID: 18654630
17.  Follicular Dendritic Cell-Specific Prion Protein (PrPc) Expression Alone Is Sufficient to Sustain Prion Infection in the Spleen 
PLoS Pathogens  2011;7(12):e1002402.
Prion diseases are characterised by the accumulation of PrPSc, an abnormally folded isoform of the cellular prion protein (PrPC), in affected tissues. Following peripheral exposure high levels of prion-specific PrPSc accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrPC is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrPC expression was specifically “switched on” or “off” only on FDC. We show that PrPC-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrPC-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrPC expression on FDC.
Author Summary
Prion diseases are infectious neurological disorders and are considered to be caused by an abnormally folded infectious protein termed PrPSc. Soon after infection prions accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the brain where they cause damage to nerve cells. Cells must express the normal cellular prion protein PrPC to become infected with prions. However, whether FDC are infected with prions or simply acquire them from other infected cells is unknown. To establish the role of FDC in prion disease PrPC expression was specifically “switched on” or “off” only on FDC. We show that PrPC-expressing FDC alone are sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrPC-expression is switched off only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues.
doi:10.1371/journal.ppat.1002402
PMCID: PMC3228802  PMID: 22144895
18.  Effect of Glycans and GPI Anchor on Strain Dependent Conformations of Scrapie Prion Protein: Improved Purifications and IR Spectra† 
Biochemistry  2011;50(21):4479-4490.
Mammalian prion diseases involve conversion of normal prion protein, PrPC, to a pathological aggregated state (PrPres). The 3D structure of PrPres is not known but infrared (IR) spectroscopy has indicated high, strain-dependent beta sheet content. PrPres molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild type PrPres with GPI- and glycan-deficient PrPres produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrPres revealed conformational differences between the 22L, ME7 and Chandler (RML) murine scrapie strains, most notably in bands attributed to beta sheets. These PrPres spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild type and anchorless 22L PrPres were nearly indistinguishable. With ME7 PrPres, modest differences between the wild type and anchorless spectra were detected, notably a ~2-cm−1 shift in an apparent beta sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrPres, at least relative to the differences observed between strains, but can subtly affect turns and certain beta sheet components. Recently reported H/D exchange analyses of anchorless PrPres preparations strongly suggested the presence of strain-dependent, solvent-inaccessible beta core structures throughout most of the C-terminal half of PrPres molecules, with no remaining alpha helix. Our present IR data provide evidence that similar core structures also comprise wild type PrPres.
doi:10.1021/bi2003907
PMCID: PMC3101284  PMID: 21539311
19.  Prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (Acinonyx jubatus). 
Journal of Virology  1990;64(5):1964-1972.
The extent and progression of exposure to feline infectious peritonitis (FIP) virus in the cheetah, Acinonyx jubatus, was monitored by a world-wide serological survey with indirect fluorescent antibody titers to coronavirus. The indirect fluorescent antibody assay was validated by Western blots, which showed that all indirect fluorescent antibody-positive cheetah sera detected both domestic cat and cheetah coronavirus structural proteins. There was a poor correlation between indirect fluorescent antibody results and the presence of coronaviruslike particles in cheetah feces, suggesting that electron microscopic detection of shed particles may not be an easily interpreted diagnostic parameter for FIP disease. Low, but verifiable (by Western blots [immunoblots]) antibody titers against coronavirus were detected in eight free-ranging cheetahs from east Africa as well as from captive cheetahs throughout the world. Of 20 North American cheetah facilities screened, 9 had cheetahs with measurable antibodies to feline coronavirus. Five facilities showed patterns of an ongoing epizootic. Retrospective FIP virus titers of an FIP outbreak in a cheetah-breeding facility in Oregon were monitored over a 5-year period and are interpreted here in terms of clinical disease progression. During that outbreak the morbidity was over 90% and the mortality was 60%, far greater than any previously reported epizootic of FIP in any cat species. Age of infection was a significant risk factor in this epizootic, with infants (less than 3 months old) displaying significantly higher risk for mortality than subadults or adults. Based upon these observations, empirical generalizations are drawn which address epidemiologic concerns for cheetahs in the context of this lethal infectious agent.
Images
PMCID: PMC249350  PMID: 2157864
20.  Strain Specific Resistance to Murine Scrapie Associated with a Naturally Occurring Human Prion Protein Polymorphism at Residue 171 
PLoS Pathogens  2011;7(9):e1002275.
Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.
Author Summary
Transmissible spongiform encephalopathies (TSE) or prion diseases are infectious fatal neurological diseases that affect many mammals, including humans. In these diseases a misfolded form of host prion protein (PrP) leads to brain degeneration and death. The genetic code of PrP in individual animals or humans has minor variations, which in some cases are associated with altered susceptibility to disease. In humans a variation at residue 171 (N171S) has been found in people mainly of African descent. However, due to the low incidence of the variation and difficult accessibility of these individuals, studies of prion diseases in these populations have not been carried out. Therefore, to create a laboratory animal model to study the effect of this variation on prion diseases, we generated transgenic mice expressing the mouse version of the human PrP variation at residue 171. We then studied the susceptibility of these mice to 4 strains of mouse-adapted scrapie. In our experiments these transgenic mice were uniquely resistant to two scrapie strains, but showed high sensitivity to two others. This resistance appeared to be related to a slow or inefficient generation of the aggregated disease-associated form of PrP in these mice, and was not duplicated using in vitro assays. In summary, transgenic mice expressing this variant PrP provide an interesting model to study differences among prion strains and their interactions with PrP in vivo.
doi:10.1371/journal.ppat.1002275
PMCID: PMC3182929  PMID: 21980292
21.  Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein 
PLoS ONE  2010;5(8):e12351.
Background
The accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.
Methodology/Principal Finding
In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.
Conclusions/Significance
Cofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.
doi:10.1371/journal.pone.0012351
PMCID: PMC2925953  PMID: 20808809
22.  Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity 
Background
Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution.
Findings
Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication.
Conclusions
This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels.
doi:10.1186/1750-1326-7-18
PMCID: PMC3355018  PMID: 22534096
Prion protein; Prion infectivity; Prion disease; Protease resistance; Subcellular localisation; Fractionation
23.  Unusual cerebral vascular prion protein amyloid distribution in scrapie-infected transgenic mice expressing anchorless prion protein 
Background
In some prion diseases, misfolded aggregated protease-resistant prion protein (PrPres) is found in brain as amyloid, which can cause cerebral amyloid angiopathy. Small diffusible precursors of PrPres amyloid might flow with brain interstitial fluid (ISF), possibly accounting for the perivascular and intravascular distribution of PrPres amyloid. We previously reported that PrPres amyloid in scrapie-infected transgenic mice appeared to delay clearance of microinjected brain ISF tracer molecules.
Results
Here we studied distribution of PrPres amyloid on capillaries, arteries and veins to test whether vascular specificity of PrPres corresponded to distribution of ISF tracer molecules. To distinguish PrPres-positive arteries from veins and capillaries, scrapie-infected mouse brains were studied by immunodetection of alpha smooth muscle actin. ISF was studied using fluorescein-labeled ovalbumin microinjected into brain as a tracer. In infected preclinical or clinical mice, PrPres was found mostly on capillaries (73-78%). Lower levels were found on arteries (11-14%) and veins (11-13%). Compared to PrPres, ISF tracer was found at higher levels on capillaries (96-97%), and the remaining tracer was found at a skewed ratio of 4 to 1 on arteries and veins respectively.
Conclusions
PrPres association with blood vessels suggested that ISF flow might transport diffusible PrPres precursor molecules to perivascular sites. However, the different vascular specificity of PrPres and ISF tracer indicated that ISF flow did not alone control PrPres dissemination. Possibly blood vessel basement membrane (BM) components, such as glucosaminoglycans, might concentrate small PrPres aggregates and serve as scaffolds for PrP conversion on multiple vessel types.
doi:10.1186/2051-5960-1-25
PMCID: PMC3893542  PMID: 24252347
Brain interstitial fluid; Cerebral amyloid angiopathy; Prion; Glycophosphatidylinositol anchor; Basement membrane
24.  Regulating Factors of PrPres Glycosylation in Creutzfeldt-Jakob Disease - Implications for the Dissemination and the Diagnosis of Human Prion Strains 
PLoS ONE  2008;3(7):e2786.
Objective
The glycoprofile of pathological prion protein (PrPres) is widely used as a diagnosis marker in Creutzfeldt-Jakob disease (CJD) and is thought to vary in a strain-specific manner. However, that the same glycoprofile of PrPres always accumulates in the whole brain of one individual has been questioned. We aimed to determine whether and how PrPres glycosylation is regulated in the brain of patients with sporadic and variant Creutzfeldt-Jakob disease.
Methods
PrPres glycoprofiles in four brain regions from 134 patients with sporadic or variant CJD were analyzed as a function of the genotype at codon 129 of PRNP and the Western blot type of PrPres.
Results
The regional distribution of PrPres glycoforms within one individual was heterogeneous in sporadic but not in variant CJD. PrPres glycoforms ratio significantly correlated with the genotype at codon 129 of the prion protein gene and the Western blot type of PrPres in a region-specific manner. In some cases of sCJD, the glycoprofile of thalamic PrPres was undistinguishable from that observed in variant CJD.
Interpretation
Regulations leading to variations of PrPres pattern between brain regions in sCJD patients, involving host genotype and Western blot type of PrPres may contribute to the specific brain targeting of prion strains and have direct implications for the diagnosis of the different forms of CJD.
doi:10.1371/journal.pone.0002786
PMCID: PMC2464735  PMID: 18665216
25.  Intraepithelial and Interstitial Deposition of Pathological Prion Protein in Kidneys of Scrapie-Affected Sheep 
PLoS ONE  2007;2(9):e859.
Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.
doi:10.1371/journal.pone.0000859
PMCID: PMC1964536  PMID: 17848990

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