Longitudinal detection of prion infection in preclinical sheep blood samples compared using 3 assays.

ABSTRACT: Variant Creutzfeldt-Jakob disease (vCJD) is a devastating disease caused by transmission of bovine spongiform encephalopathy to humans. Although vCJD cases are now rare, evidence from appendix surveys suggests that a small proportion of the United Kingdom population may be infected without showing signs of disease. These "silent" carriers could present a risk of iatrogenic vCJD transmission through medical procedures or blood/organ donation, and currently there are no validated tests to identify infected asymptomatic individuals using easily accessible samples. To address this issue, we evaluated the performance of 3 blood-based assays in a blinded study, using longitudinal sample series from a well-established large animal model of vCJD. The assays rely on amplification of misfolded prion protein (PrPSc; a marker of prion infection) and include real-time quaking-induced conversion (RT-QuIC), and 2 versions of protein misfolding cyclic amplification (PMCA). Although diagnostic sensitivity was higher for both PMCA assays (100%) than RT-QuIC (61%), all 3 assays detected prion infection in blood samples collected 26 months before the onset of clinical signs and gave no false-positive results. Parallel estimation of blood prion infectivity titers in a sensitive transgenic mouse line showed positive correlation of infectivity with PrPSc detection by the assays, suggesting that they are suitable for detection of asymptomatic vCJD infection in the human population. This study represents, to our knowledge, the largest comparison to date of preclinical prion detection in blood samples from a relevant animal model. The outcomes will guide efforts to improve early detection of prion disease and reduce infection risks in humans.

Lack of prion transmission barrier in human PrP transgenic Drosophila.

While animal prion diseases are a threat to human health, their zoonotic potential is generally inefficient because of interspecies prion transmission barriers. New animal models are required to provide an understanding of these prion transmission barriers and to assess the zoonotic potential of animal prion diseases. To address this goal, we generated Drosophila transgenic for human or nonhuman primate prion protein (PrP) and determined their susceptibility to known pathogenic prion diseases, namely varient Creutzfeldt-Jakob disease (vCJD) and classical bovine spongiform encephalopathy (BSE), and that with unknown pathogenic potential, namely chronic wasting disease (CWD). Adult Drosophila transgenic for M129 or V129 human PrP or nonhuman primate PrP developed a neurotoxic phenotype and showed an accelerated loss of survival after exposure to vCJD, classical BSE, or CWD prions at the larval stage. vCJD prion strain identity was retained after passage in both M129 and V129 human PrP Drosophila. All of the primate PrP fly lines accumulated prion seeding activity and concomitantly developed a neurotoxic phenotype, generally including accelerated loss of survival, after exposure to CWD prions derived from different cervid species, including North American white-tailed deer and muntjac, and European reindeer and moose. These novel studies show that primate PrP transgenic Drosophila lack known prion transmission barriers since, in mammalian hosts, V129 human PrP is associated with severe resistance to classical BSE prions, while both human and cynomolgus macaque PrP are associated with resistance to CWD prions. Significantly, our data suggest that interspecies differences in the amino acid sequence of PrP may not be a principal determinant of the prion transmission barrier.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy.

The role of the glycosylation status of PrPC in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrPC. Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrPC-expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrPC is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Variation in the prion protein gene (PRNP) open reading frame sequence in French cervids.

The recent emergence of chronic wasting disease (CWD) in Europe has become a new public health risk for monitoring of wild and farmed cervids. This disease, due to prions, has proliferated in North America in a contagious manner. In several mammalian species, polymorphisms in the prion protein gene (PRNP) play a crucial role in the susceptibility to prions and their spread. To obtain a reliable picture of the distribution of PRNP polymorphisms in the two most common cervid species in France, we sequenced the open reading frame (ORF) of this gene in 2114 animals, 1116 roe deer (Capreolus capreolus) and 998 red deer (Cervus elaphus). Selection criteria such as historical origin, spatial distribution and sex ratio have been integrated to establish this sample collection. Except for one heterozygous animal with a non-synonymous mutation at codon 37 (G37A), all the 1116 French roe deer were monomorphic. Red deer showed greater variation with two non-synonymous substitutions (T98A; Q226E), three synonymous substitutions (codons 21, 78 and 136) and a new 24pb deletion (Δ69-77). We found significant regional variations between French regions in the frequency of the identified substitutions. After cloning of the PRNP ORF from animals presenting multiple non-synonymous polymorphisms, we identified six haplotypes and obtained a total of twelve genotypes. As in other European countries, we highlighted the apparent homogeneity of PRNP in the French roe deer and the existence of a greater diversity in the red deer. These results were in line with European phylogeographic studies on these two species.

Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Correction: Site-specific analysis of N-glycans from different sheep prion strains.

[This corrects the article DOI: 10.1371/journal.ppat.1009232.].

Site-specific analysis of N-glycans from different sheep prion strains.

Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian species, including humans. During the course of the disease, the abnormally folded scrapie prion protein (PrPSc) accumulates in the central nervous system where it causes neurodegeneration. In prion disorders, the diverse spectrum of illnesses exists because of the presence of different isoforms of PrPSc where they occupy distinct conformational states called strains. Strains are biochemically distinguished by a characteristic three-band immunoblot pattern, defined by differences in the occupancy of two glycosylation sites on the prion protein (PrP). Characterization of the exact N-glycan structures attached on either PrPC or PrPSc is lacking. Here we report the characterization and comparison of N-glycans from two different sheep prion strains. PrPSc from both strains was isolated from brain tissue and enzymatically digested with trypsin. By using liquid chromatography coupled to electrospray mass spectrometry, a site-specific analysis was performed. A total of 100 structures were detected on both glycosylation sites. The N-glycan profile was shown to be similar to the one on mouse PrP, however, with additional 40 structures reported. The results presented here show no major differences in glycan composition, suggesting that glycans may not be responsible for the differences in the two analyzed prion strains.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Prion disease modelled in Drosophila.

Prion diseases are fatal neurodegenerative conditions of humans and various vertebrate species that are transmissible between individuals of the same or different species. A novel infectious moiety referred to as a prion is considered responsible for transmission of these conditions. Prion replication is believed to be the cause of the neurotoxicity that arises during prion disease pathogenesis. The prion hypothesis predicts that the transmissible prion agent consists of PrPSc, which is comprised of aggregated misfolded conformers of the normal host protein PrPC. It is important to understand the biology of transmissible prions and to identify genetic modifiers of prion-induced neurotoxicity. This information will underpin the development of therapeutic and control strategies for human and animal prion diseases. The most reliable method to detect prion infectivity is by in vivo transmission in a suitable experimental host, which to date have been mammalian species. Current prion bioassays are slow, cumbersome and relatively insensitive to low titres of prion infectivity, and do not lend themselves to rapid genetic analysis of prion disease. Here, we provide an overview of our novel studies that have led to the establishment of Drosophila melanogaster, a genetically well-defined invertebrate host, as a sensitive, versatile and economically viable animal model for the detection of mammalian prion infectivity and genetic modifiers of prion-induced toxicity.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie.

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system.

The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. Here we have tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so we used prion protein (PrP) transgenic Drosophila that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was downregulated in prion-exposed PrP Drosophila. We demonstrated that RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas overexpression mitigated toxicity. Strikingly, prion seeding activity in variant Creutzfeldt-Jakob disease prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 overexpression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.

A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila.

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10-12 dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 106-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 105-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.

Host prion protein expression levels impact prion tropism for the spleen.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.

RNA editing alterations define manifestation of prion diseases.

Prion diseases are fatal neurodegenerative disorders caused by misfolding of the normal prion protein into an infectious cellular pathogen. Clinically characterized by rapidly progressive dementia and accounting for 85% of human prion disease cases, sporadic Creutzfeldt-Jakob disease (sCJD) is the prevalent human prion disease. Although sCJD neuropathological hallmarks are well-known, associated molecular alterations are elusive due to rapid progression and absence of preclinical stages. To investigate transcriptome alterations during disease progression, we utilized tg340-PRNP129MM mice infected with postmortem material from sCJD patients of the most susceptible genotype (MM1 subtype), a sCJD model that faithfully recapitulates the molecular and pathological alterations of the human disease. Here we report that transcriptomic analyses from brain cortex in the context of disease progression, reveal epitranscriptomic alterations (specifically altered RNA edited pathway profiles, eg., ER stress, lysosome) that are characteristic and possibly protective mainly for preclinical and clinical disease stages. Our results implicate regulatory epitranscriptomic mechanisms in prion disease neuropathogenesis, whereby RNA-editing targets in a humanized sCJD mouse model were confirmed in pathological human autopsy material.

The emergence of classical BSE from atypical/Nor98 scrapie.

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.

Porcine Prion Protein as a Paradigm of Limited Susceptibility to Prion Strain Propagation.

Although experimental transmission of bovine spongiform encephalopathy (BSE) to pigs and transgenic mice expressing pig cellular prion protein (PrPC) (porcine PrP [PoPrP]-Tg001) has been described, no natural cases of prion diseases in pig were reported. This study analyzed pig-PrPC susceptibility to different prion strains using PoPrP-Tg001 mice either as animal bioassay or as substrate for protein misfolding cyclic amplification (PMCA). A panel of isolates representatives of different prion strains was selected, including classic and atypical/Nor98 scrapie, atypical-BSE, rodent scrapie, human Creutzfeldt-Jakob-disease and classic BSE from different species. Bioassay proved that PoPrP-Tg001-mice were susceptible only to the classic BSE agent, and PMCA results indicate that only classic BSE can convert pig-PrPC into scrapie-type PrP (PrPSc), independently of the species origin. Therefore, conformational flexibility constraints associated with pig-PrP would limit the number of permissible PrPSc conformations compatible with pig-PrPC, thus suggesting that pig-PrPC may constitute a paradigm of low conformational flexibility that could confer high resistance to the diversity of prion strains.

Co-invalidation of Prnp and Sprn in FVB/N mice affects reproductive performances and highlight complex biological relationship between PrP and Shadoo.

Shadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes' knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process.

Wide distribution of prion infectivity in the peripheral tissues of vCJD and sCJD patients.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the commonest human prion disease, occurring most likely as the consequence of spontaneous formation of abnormal prion protein in the central nervous system (CNS). Variant Creutzfeldt-Jakob disease (vCJD) is an acquired prion disease that was first identified in 1996. In marked contrast to vCJD, previous investigations in sCJD revealed either inconsistent levels or an absence of PrPSc in peripheral tissues. These findings contributed to the consensus that risks of transmitting sCJD as a consequence of non-CNS invasive clinical procedures were low. In this study, we systematically measured prion infectivity levels in CNS and peripheral tissues collected from vCJD and sCJD patients. Unexpectedly, prion infectivity was detected in a wide variety of peripheral tissues in sCJD cases. Although the sCJD infectivity levels varied unpredictably in the tissues sampled and between patients, these findings could impact on our perception of the possible transmission risks associated with sCJD.