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.

Allelic Interference in Prion Replication Is Modulated by the Convertibility of the Interfering PrPC and Other Host-Specific Factors.

Early studies in transgenic mouse lines have shown that the coexpression of endogenous murine prion protein (PrPC) and transgenic PrPC from another species either inhibits or allows the propagation of prions, depending on the infecting prion strain and interacting protein species. The way whereby this phenomenon, so-called "interference," is modulated remains to be determined. In this study, different transgenic mouse lines were crossbred to produce mice coexpressing bovine and porcine PrPC, bovine and murine PrPC, or murine and porcine PrPC These animals and their respective hemizygous controls were inoculated with several prion strains from different sources (cattle, mice, and pigs) to examine the effects of the simultaneous presence of PrPC from two different species. Our results indicate interference with the infection process, manifested as extended survival times and reduced attack rates. The interference with the infectious process was reduced or absent when the potentiality interfering PrPC species was efficiently converted by the inoculated agent. However, the propagation of the endogenous murine PrPSc was favored, allowing us to speculate that host-specific factors may disturb the interference caused by the coexpression of an exogenous second PrPCIMPORTANCE Prion propagation can be interfered with by the expression of a second prion protein in the host. In the present study, we investigated prion propagation in a host expressing two different prion protein genes. Our findings indicate that the ability of the second prion protein to interfere with prion propagation is related to the transmissibility of the prion in the host expressing only the interfering prion protein. The interference detected occurs in a prion strain-dependent manner. Interestingly, a bias favoring the propagation of the murine PrP allele has been observed. These results open the door to future studies in order to determine the role of host factors other than the PrP amino acid sequence in the interference in prion propagation.

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.

Prion infection, transmission, and cytopathology modeled in a low-biohazard human cell line.

Transmission of prion infectivity to susceptible murine cell lines has simplified prion titration assays and has greatly reduced the need for animal experimentation. However, murine cell models suffer from technical and biological constraints. Human cell lines might be more useful, but they are much more biohazardous and are often poorly infectible. Here, we describe the human clonal cell line hovS, which lacks the human PRNP gene and expresses instead the ovine PRNP VRQ allele. HovS cells were highly susceptible to the PG127 strain of sheep-derived murine prions, reaching up to 90% infected cells in any given culture and were maintained in a continuous infected state for at least 14 passages. Infected hovS cells produced proteinase K-resistant prion protein (PrPSc), pelletable PrP aggregates, and bona fide infectious prions capable of infecting further generations of naïve hovS cells and mice expressing the VRQ allelic variant of ovine PrPC Infection in hovS led to prominent cytopathic vacuolation akin to the spongiform changes observed in individuals suffering from prion diseases. In addition to expanding the toolbox for prion research to human experimental genetics, the hovS cell line provides a human-derived system that does not require human prions. Hence, the manipulation of scrapie-infected hovS cells may present fewer biosafety hazards than that of genuine human prions.

Prions from Sporadic Creutzfeldt-Jakob Disease Patients Propagate as Strain Mixtures.

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently classified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K-digested abnormal prion protein (PrPres) isoform identified by Western blotting (type 1 or type 2). Converging evidence led to the view that MM/MV1, VV/MV2, and VV1 and MM2 sCJD cases are caused by distinct prion strains. However, in a significant proportion of sCJD patients, both type 1 and type 2 PrPres were reported to accumulate in the brain, which raised questions about the diversity of sCJD prion strains and the coexistence of two prion strains in the same patient. In this study, a panel of sCJD brain isolates (n = 29) that displayed either a single or mixed type 1/type 2 PrPres were transmitted into human-PrP-expressing mice (tgHu). These bioassays demonstrated that two distinct prion strains (M1CJD and V2CJD) were associated with the development of sCJD in MM1/MV1 and VV2/MV2 patients. However, in about 35% of the investigated VV and MV cases, transmission results were consistent with the presence of both M1CJD and V2CJD strains, including in patients who displayed a "pure" type 1 or type 2 PrPres The use of a highly sensitive prion in vitro amplification technique that specifically probes the V2CJD strain revealed the presence of the V2CJD prion in more than 80% of the investigated isolates, including isolates that propagated as a pure M1CJD strain in tgHu. These results demonstrate that at least two sCJD prion strains can be present in a single patient.IMPORTANCE sCJD occurrence is currently assumed to result from spontaneous and stochastic formation of a misfolded PrP nucleus in the brains of affected patients. This original nucleus then recruits and converts nascent PrPC into PrPSc, leading to the propagation of prions in the patient's brain. Our study demonstrates the coexistence of two prion strains in the brains of a majority of the 23 sCJD patients investigated. The relative proportion of these sCJD strains varied both between patients and between brain areas in a single patient. These findings strongly support the view that the replication of an sCJD prion strain in the brain of a patient can result in the propagation of different prion strain subpopulations. Beyond its conceptual importance for our understanding of prion strain properties and evolution, the sCJD strain mixture phenomenon and its frequency among patients have important implications for the development of therapeutic strategies for prion diseases.

Radical Change in Zoonotic Abilities of Atypical BSE Prion Strains as Evidenced by Crossing of Sheep Species Barrier in Transgenic Mice.

Classical bovine spongiform encephalopathy (BSE) is the only zoonotic prion disease described to date. Although the zoonotic potential of atypical BSE prions have been partially studied, an extensive analysis is still needed. We conducted a systematic study by inoculating atypical BSE isolates from different countries in Europe into transgenic mice overexpressing human prion protein (PrP): TgMet129, TgMet/Val129, and TgVal129. L-type BSE showed a higher zoonotic potential in TgMet129 mice than classical BSE, whereas Val129-PrP variant was a strong molecular protector against L-type BSE prions, even in heterozygosis. H-type BSE could not be transmitted to any of the mice. We also adapted 1 H- and 1 L-type BSE isolate to sheep-PrP transgenic mice and inoculated them into human-PrP transgenic mice. Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet129 and TgVal129, bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.

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.

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.

Infectivity in bone marrow from sporadic CJD patients.

Prion infectivity was recently identified in the blood of both sporadic and variant Creutzfeldt-Jakob disease (CJD) patients. In variant CJD (vCJD), the widespread distribution of prions in peripheral tissues of both asymptomatic and symptomatic patients is likely to explain the occurrence of the observed prionaemia. However, in sporadic CJD (sCJD), prion infectivity is described to be located principally in the central nervous system. In this study, we investigated the presence of prion infectivity in bone marrow collected after death in patients affected with different sCJD agents. Bioassays in transgenic mice expressing the human prion protein revealed the presence of unexpectedly high levels of infectivity in the bone marrow from seven out of eight sCJD cases. These findings may explain the presence of blood-borne infectivity in sCJD patients. They also suggest that the distribution of prion infectivity in peripheral tissues in sCJD patients could be wider than currently believed, with potential implications for the iatrogenic transmission risk of this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Classical scrapie transmission in ARR/ARR genotype sheep.

The ARR allele is considered to provide a very strong resistance against classical scrapie infection in sheep. In this study, we report the occurrence of clinical transmissible spongiform encephalopathy in ARR/ARR sheep, following their inoculation by the intracerebral route with a classical scrapie isolate. On first passage, the disease displayed an incomplete attack rate transmission, with incubation periods exceeding 6 years. On second passage, the obtained prion did not display better abilities to propagate than the original isolate. These transmission results contrasted with the 100 % attack rate and the short incubation periods observed in ARQ/ARQ sheep challenged with the same isolate. These data confirm that ARR/ARR sheep cannot be considered to be fully resistant to classical scrapie. However, they also support the contention that classical scrapie has a very limited capacity to transmit and adapt to ARR/ARR sheep.

Distribution and Quantitative Estimates of Variant Creutzfeldt-Jakob Disease Prions in Tissues of Clinical and Asymptomatic Patients.

In the United-Kingdom, ≈1 of 2,000 persons could be infected with variant Creutzfeldt-Jakob disease (vCJD). Therefore, risk of transmission of vCJD by medical procedures remains a major concern for public health authorities. In this study, we used in vitro amplification of prions by protein misfolding cyclic amplification (PMCA) to estimate distribution and level of the vCJD agent in 21 tissues from 4 patients who died of clinical vCJD and from 1 asymptomatic person with vCJD. PMCA identified major levels of vCJD prions in a range of tissues, including liver, salivary gland, kidney, lung, and bone marrow. Bioassays confirmed that the quantitative estimate of levels of vCJD prion accumulation provided by PMCA are indicative of vCJD infectivity levels in tissues. Findings provide critical data for the design of measures to minimize risk for iatrogenic transmission of vCJD.

Mononucleated Blood Cell Populations Display Different Abilities To Transmit Prion Disease by the Transfusion Route.

UNLABELLED: Previous experiments carried out in a sheep scrapie model demonstrated that the transfusion of 200 µl of prion-infected whole blood has an apparent 100% efficacy for disease transmission. These experiments also indicated that, despite the apparent low infectious titer, the intravenous administration of white blood cells (WBC) resulted in efficient disease transmission. In the study presented here, using the same transmissible spongiform encephalopathy (TSE) animal model, our aim was to determine the minimal number of white blood cells and the specific abilities of mononucleated cell populations to transmit scrapie by the transfusion route. Our results confirmed that the transfusion of 100 µl, but not 10 µl, of fresh whole blood collected in asymptomatic scrapie-infected donor sheep can transmit the disease. The data also show that the intravenous administration of 10(5) WBCs is sufficient to cause scrapie in recipient sheep. Cell-sorted CD45R(+) (predominantly B lymphocytes), CD4(+)/CD8(+) (T lymphocytes), and CD14(+) (monocytes/macrophages) blood cell subpopulations all were shown to contain prion infectivity by bioassays in ovine PrP transgenic mice. However, while the intravenous administration of 10(6) CD45(+) or CD4(+)/8(+) living cells was able to transmit the disease, similar numbers of CD14(+) cells failed to infect the recipients. These data support the contention that mononucleated blood cell populations display different abilities to transmit TSE by the transfusion route. They also represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine. IMPORTANCE: Interindividual variant Creutzfeldt-Jakob disease (vCJD) transmission through blood and blood-derived products is considered a major public health issue in transfusion medicine. Over the last decade, TSE in sheep has emerged as a relevant model for assessing the blood-borne vCJD transmission risk. In this study, using a sheep TSE model, we characterized the ability of different peripheral blood mononucleated cell populations to infect TSE-free recipients by the transfusion route. Our results indicate that as little as 10(5) WBC and 100 µl of blood collected from asymptomatic scrapie infected animals can transmit the disease. They also demonstrate unambiguously that peripheral blood mononuclear cell subpopulations display dramatically different abilities to transmit the disease. These data represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine.

Evidence for zoonotic potential of ovine scrapie prions.

Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.

White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

Preclinical detection of variant CJD and BSE prions in blood.

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.

PrP expression level and sensitivity to prion infection.

Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer.

Detection of infectivity in blood of persons with variant and sporadic Creutzfeldt-Jakob disease.

We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies.

Genetic resistance to scrapie infection in experimentally challenged goats.

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M142, N/D146, S/D146, R/Q211, and Q/K222) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I142R154R211Q222) goats and homozygous and/or heterozygous I/M142, R/H154, R/Q211, and Q/K222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H154, Q211, and K222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H154 and Q211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K222 and a small proportion of homozygous K222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.

The limits of test-based scrapie eradication programs in goats.

Small ruminant post-mortem testing programs were initially designed for monitoring the prevalence of prion disease. They are now considered as a potential alternative to genetic selection for eradicating/controlling classical scrapie at population level. If such policy should be implemented, its success would be crucially dependent on the efficiency of the surveillance system used to identify infected flocks. In this study, we first determined the performance of post-mortem classical scrapie detection in eight naturally affected goat herds (total n = 1961 animals) according to the age at culling. These results provided us with necessary parameters to estimate, through a Monte Carlo simulation model, the performance of scrapie detection in a commercial population. According to this model, whatever the number of tests performed, post mortem surveillance will have limited success in identifying infected herds. These data support the contention that scrapie eradication programs relying solely on post mortem testing in goats will probably fail. Considering the epidemiological and pathological similarities of scrapie in sheep and goats, the efficiency of scrapie surveillance in both species is likely to be similar.