Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge.

After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrPBSE in peripheral nervous tissues during the first eight months post-inoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrPBSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrPBSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.

Classical BSE prions emerge from asymptomatic pigs challenged with atypical/Nor98 scrapie.

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrPSc accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8-9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrPSc accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.

Prion strains associated with iatrogenic CJD in French and UK human growth hormone recipients.

Treatment with human pituitary-derived growth hormone (hGH) was responsible for a significant proportion of iatrogenic Creutzfeldt-Jakob disease (iCJD) cases. France and the UK experienced the largest case numbers of hGH-iCJD, with 122 and 81 cases respectively. Differences in the frequency of the three PRNP codon 129 polymorphisms (MM, MV and VV) and the estimated incubation periods associated with each of these genotypes in the French and the UK hGH-iCJD cohorts led to the suggestion that the prion strains responsible for these two hGH-iCJD cohorts were different. In this study, we characterized the prion strains responsible for hGH-iCJD cases originating from UK (n = 11) and France (n = 11) using human PrP expressing mouse models. The cases included PRNP MM, MV and VV genotypes from both countries. UK and French sporadic CJD (sCJD) cases were included as controls. The prion strains identified following inoculation with hGH-iCJD homogenates corresponded to the two most frequently observed sCJD prion strains (M1CJD and V2CJD). However, in clear contradiction to the initial hypothesis, the prion strains that were identified in the UK and the French hGH-iCJD cases were not radically different. In the vast majority of the cases originating from both countries, the V2CJD strain or a mixture of M1CJD + V2CJD strains were identified. These data strongly support the contention that the differences in the epidemiological and genetic profiles observed in the UK and France hGH-iCJD cohorts cannot be attributed only to the transmission of different prion strains.

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.

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.

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.

Absence of classical and atypical (H- and L-) BSE infectivity in the blood of bovines in the clinical end stage of disease as confirmed by intraspecies blood transfusion.

While the presence of bovine spongiform encephalopathy (BSE) infectivity in the blood of clinically affected sheep has been proven by intraspecies blood-transfusion experiments, this question has remained open in the case of BSE-affected cattle. Although the absence of infectivity can be anticipated from the restriction of the agent to neuronal tissues in this species, evidence for this was still lacking. This particularly concerns the production and use of medicinal products and other applications containing bovine blood or preparations thereof. We therefore performed a blood-transfusion experiment from cattle in the clinical end stage of disease after experimental challenge with either classical (C-BSE) or atypical (H- and l-) BSE into calves at 4-6 months of age. The animals were kept in a free-ranging group for 10 years. Starting from 24 months post-transfusion, a thorough clinical examination was performed every 6 weeks in order to detect early symptoms of a BSE infection. Throughout the experiment, the clinical picture of all animals gave no indication of a BSE infection. Upon necropsy, the brainstem samples were analysed by BSE rapid test as well as by the highly sensitive Protein Misfolding Cyclic Amplification (PMCA), all with negative results. These results add resilient data to confirm the absence of BSE infectivity in the donor blood collected from C-, H- and l-BSE-affected cattle even in the final clinical phase of the disease. This finding has important implications for the risk assessment of bovine blood and blood products in the production of medicinal products and other preparations.

Bovine adapted transmissible mink encephalopathy is similar to L-BSE after passage through sheep with the VRQ/VRQ genotype but not VRQ/ARQ.

BACKGROUND: Transmissible mink encephalopathy (TME) is a fatal neurologic disease of farmed mink. Evidence indicates that TME and L-BSE are similar and may be linked in some outbreaks of TME. We previously transmitted bovine adapted TME (bTME) to sheep. The present study compared ovine passaged bTME (o-bTME) to C-BSE and L-BSE in transgenic mice expressing wild type bovine prion protein (TgBovXV). To directly compare the transmission efficiency of all prion strains in this study, we considered the attack rates and mean incubation periods. Additional methods for strain comparison were utilized including lesion profiles, fibril stability, and western blotting. RESULTS: Sheep donor genotype elicited variable disease phenotypes in bovinized mice. Inoculum derived from a sheep with the VRQ/VRQ genotype (o-bTMEVV) resulted in an attack rate, incubation period, western blot profile, and neuropathology most similar to bTME and L-BSE. Conversely, donor material from a sheep with the VRQ/ARQ genotype (o-bTMEAV) elicited a phenotype distinct from o-bTMEVV, bTME and L-BSE. The TSE with the highest transmission efficiency in bovinized mice was L-BSE. The tendency to efficiently transmit to TgBovXV mice decreased in the order bTME, C-BSE, o-bTMEVV, and o-bTMEAV. The transmission efficiency of L-BSE was approximately 1.3 times higher than o-bTMEVV and 3.2 times higher than o-bTMEAV. CONCLUSIONS: Our findings provide insight on how sheep host genotype modulates strain genesis and influences interspecies transmission characteristics. Given that the transmission efficiencies of L-BSE and bTME are higher than C-BSE, coupled with previous reports of L-BSE transmission to mice expressing the human prion protein, continued monitoring for atypical BSE is advisable in order to prevent occurrences of interspecies transmission that may affect humans or other species.

Characterization of goat prions demonstrates geographical variation of scrapie strains in Europe and reveals the composite nature of prion strains.

Bovine Spongiform Encephalopathy (BSE) is the only animal prion which has been recognized as a zoonotic agent so far. The identification of BSE in two goats raised the need to reliably identify BSE in small ruminants. However, our understanding of scrapie strain diversity in small ruminants remains ill-defined, thus limiting the accuracy of BSE surveillance and spreading fear that BSE might lurk unrecognized in goats. We investigated prion strain diversity in a large panel of European goats by a novel experimental approach that, instead of assessing the neuropathological profile after serial transmissions in a single animal model, was based on the direct interaction of prion isolates with several recipient rodent models expressing small ruminants or heterologous prion proteins. The findings show that the biological properties of scrapie isolates display different patterns of geographical distribution in Europe and suggest that goat BSE could be reliably discriminated from a wide range of biologically and geographically diverse goat prion isolates. Finally, most field prion isolates showed composite strain features, with discrete strain components or sub-strains being present in different proportions in individual goats or tissues. This has important implications for understanding the nature and evolution of scrapie strains and their transmissibility to other species, including humans.

Eliminating transmissibility of bovine spongiform encephalopathy by dry-heat treatment.

Bovine spongiform encephalopathy (BSE) prion is more resistant to heat inactivation compared to other prions, but the effect of heat inactivation has been reported to differ depending on the BSE-contaminated tissue state or heating type. We aimed to evaluate the secure level of inactivation of original BSE transmissibility by dry-heating. Cattle tissues affected with BSE were subjected to dry-heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C. To assess the inactivation effect, we conducted protein misfolding cyclic amplification (PMCA) and follicular dendritic cell (FDC) assays in transgenic mice expressing bovine prion protein genes. Under dry-heating at 600 °C or higher, BSE cattle tissues lost their transmissibility in transgenic mice. In contrast, transmissibility was detected in the cattle tissues treated at temperatures of 400 °C or lower through the FDC assay combined with PMCA. In this study, we confirmed that transmissibility was eliminated in BSE-affected cattle tissues by dry-heating at 600 °C or higher.

Tracking and clarifying differential traits of classical- and atypical L-type bovine spongiform encephalopathy prions after transmission from cattle to cynomolgus monkeys.

Classical- (C-) and atypical L-type bovine spongiform encephalopathy (BSE) prions cause different pathological phenotypes in cattle brains, and the disease-associated forms of each prion protein (PrPSc) has a dissimilar biochemical signature. Bovine C-BSE prions are the causative agent of variant Creutzfeldt-Jakob disease. To date, human infection with L-BSE prions has not been reported, but they can be transmitted experimentally from cows to cynomolgus monkeys (Macaca fascicularis), a non-human primate model. When transmitted to monkeys, C- and L-BSE prions induce different pathological phenotypes in the brain. However, when isolated from infected brains, the two prion proteins (PrPSc) have similar biochemical signatures (i.e., electrophoretic mobility, glycoforms, and resistance to proteinase K). Such similarities suggest the possibility that L-BSE prions alter their virulence to that of C-BSE prions during propagation in monkeys. To clarify this possibility, we conducted bioassays using inbred mice. C-BSE prions with or without propagation in monkeys were pathogenic to mice, and exhibited comparable incubation periods in secondary passage in mice. By contrast, L-BSE prions, either with or without propagation in monkeys, did not cause the disease in mice, indicating that the pathogenicity of L-BSE prions does not converge towards a C-BSE prion type in this primate model. These results suggest that, although C- and L-BSE prions propagated in cynomolgus monkeys exhibit similar biochemical PrPSc signatures and consist of the monkey amino acid sequence, the two prions maintain strain-specific conformations of PrPSc in which they encipher and retain unique pathogenic traits.

BSE infectivity survives burial for five years with only limited spread.

The carcasses of animals infected with bovine spongiform encephalopathy (BSE), scrapie or chronic wasting disease (CWD) that remain in the environment (exposed or buried) may continue to act as reservoirs of infectivity. We conducted two experiments under near-field conditions to investigate the survival and dissemination of BSE infectivity after burial in a clay or sandy soil. BSE infectivity was either contained within a bovine skull or buried as an uncontained bolus of BSE-infected brain. Throughout the five-year period of the experiment, BSE infectivity was recovered in similar amounts from heads exhumed annually from both types of soil. Very low levels of infectivity were detected in the soil immediately surrounding the heads, but not in samples remote from them. Similarly, there was no evidence of significant lateral movement of infectivity from the buried bolus over 4 years although there was a little vertical movement in both directions. However, bioassay analysis of limited numbers of samples of rain water that had drained through the bolus clay lysimeter indicated that infectivity was present in filtrates. sPMCA analysis also detected low levels of PrPSc in the filtrates up to 25 months following burial, raising the concern that leakage of infectivity into ground water could occur. We conclude that transmissible spongiform encephalopathy infectivity is likely to survive burial for long periods of time, but not to migrate far from the site of burial unless a vector or rain water drainage transports it. Risk assessments of contaminated sites should take these findings into account.

Low levels of classical BSE infectivity in rendered fat tissue.

BSE infectivity in mesentery fat is most likely associated with embedded nervous tissue. To prove this mesentery containing celiac ganglion was taken from oral BSE infected cattle in different stages of the disease and from one control animal. Fat was rendered according to standard tallow production methods and the prion infectivity therein analysed in transgenic mouse bioassay. Rendered fat of the clinical animal revealed low infectivity levels, whereas preclinical and control animals remained negative. This study, although not representative, provides a proof of principle, indicating the potential contamination of melted mesenteric fat by embedded nervous structures during standard tallow production.

Molecular characterisation of atypical BSE prions by mass spectrometry and changes following transmission to sheep and transgenic mouse models.

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Detection of PrPres in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent.

Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrPres accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrPres positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain.

The zoonotic potential of animal prion diseases.

Bovine spongiform encephalopathy (BSE) is the only animal prion disease that has been demonstrated to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans. The link between BSE and vCJD was established by careful surveillance, epidemiologic investigations, and experimental studies using in vivo and in vitro models of cross-species transmission. Similar approaches have been used to assess the zoonotic potential of other animal prion diseases, including atypical forms identified through active surveillance. There is no epidemiologic evidence that classical or atypical scrapie, atypical forms of BSE, or chronic wasting disease (CWD) is associated with human prion disease, but the limitations of the epidemiologic data should be taken into account when interpreting these results. Transmission experiments in nonhuman primates and human PrP transgenic mice suggest that classic scrapie, L-type atypical BSE (L-BSE), and CWD may have zoonotic potential, which for L-BSE appears to be equal to or greater than that of classic BSE. The results of in vitro conversion assays to analyze the human transmission barrier correlate well with the in vivo data. However, it is still difficult to predict the likelihood that an animal prion disease will transmit to humans under conditions of field exposure from the results of in vivo or in vitro experiments. This emphasizes the importance of continuing systematic surveillance for both human and animal prion diseases in identifying zoonotic transmission of diseases other than classic BSE.

Clinical, pathological, and molecular features of classical and L-type atypical-BSE in goats.

Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health.

Presumptive BSE cases with an aberrant prion protein phenotype in Switzerland, 2011: Lack of prion disease in experimentally inoculated cattle and bovine prion protein transgenic mice.

Bovine spongiform encephalopathy (BSE) is caused by different prion strains that are discriminated by the molecular characteristics of the pathological prion protein. In 2011, Switzerland reported two presumptive cases of BSE in cattle with a prion protein phenotype different from previously described strains, and it was unclear whether these findings were related to a transmissible disease and have implications on animal and public health. In this study, brain tissues of these cases were inoculated into transgenic mice expressing the bovine prion protein (BoPrP-Tg110) and into cattle. Clinical and pathological investigations as well as molecular testing did not provide evidence for the presence of BSE in the Swiss cases after two passages in BoPrP-Tg110 mice and a challenge period of 3.5 years in cattle. This lack of disease transmission suggests that the Swiss 2011 cases were not affected by a prion disease and were unrelated to the feed-born BSE epidemic.

Rapid amplification of prions from variant Creutzfeldt-Jakob disease cerebrospinal fluid.

Human prion diseases constitute a group of infectious and invariably fatal neurodegenerative disorders associated with misfolding of the prion protein. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonotic prion disease linked to oral exposure to the infectious agent that causes bovine spongiform encephalopathy (BSE) in cattle. The most recent case of definite vCJD was heterozygous (MV) at polymorphic codon 129 of the prion protein gene PRNP while all of the previous 177 definite or probable vCJD cases who underwent genetic analysis were methionine homozygous (MM). Retrospective prevalence studies conducted on lympho-reticular tissue suggest that the number of asymptomatic vCJD carriers in the United Kingdom might be around 1 in 2000 people. In addition, there have been four known cases of the transmission of vCJD infection via blood transfusion. For these reasons, a sensitive, reliable, and fast diagnostic test is currently needed. We describe a rapid and highly sensitive seeding conversion assay that detects disease-associated prion protein in the brain and cerebrospinal fluid in vCJD after 48-96 h of amplification, with 100% sensitivity and specificity. This method can amplify prions from definite, probable, and possible vCJD cases from patients who are either MM or MV at PRNP-codon 129.