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The article presents a study which described the behaviour of sheep infected by bovine spongiform encephalopathy (BSE). It compared two groups of 11 ARR (used as controls) homozygous and 9 ARQ (highly susceptible) homozygous sheep. Among the things they analyzed were the duration of resting, standing, feeding, walking and scraping activities. According to the researchers, despite their failure to find early behavioural signs of BSE in sheep, the results confirmed the significance of scraping activity in BSE infected sheep. They claim that the results are consistent with another study which found a rapid rise of the percentages of animals showing clinical signs.

The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)-infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc. [ABSTRACT FROM AUTHOR]

Because variant Creutzfeldt-Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrPSc in a myocyte subset. In naturally infected sheep, PrPSc is detectable in muscle several months before clinical disease onset. The relative amounts of PrPSc suggest a 5,000-fold lower infectivity for muscle as compared to brain. [ABSTRACT FROM AUTHOR]

Abstract Background The retina is part of the diencephalon in a peripheral location and may be involved in prion diseases. Retinal function and structural changes were assessed in naturally scrapie-affected red face Manech ewes presenting the classical signs of the disease, and clinically healthy age-matched subjects for controls. Ophthalmic examination was done prior to electroretinography (ERG), which was carried out under conditions that allowed photopic and scotopic activities to be assessed. Histomorphometry of the inner and outer retinal layers was performed post-mortem, and retinas were also examined for evidence of abnormal prion protein (PrPSc) accumulation and glial fibrillary acidic protein (GFAP) upregulation as a marker of gliosis. Scrapie status was determined by examination of brain tissue Results Ocular reflexes and ophthalmoscopy did not reveal any difference between scrapie affected and control animals. Although the light-and dark-adapted ERG responses of both rod-and cone-mediated functions had a similar waveform in scrapie-affected and control sheep, a significant reduction in the amplitude of the ERG a-and b-waves was observed in affected animals compared to controls. These functional alterations were correlated with a substantial loss of cells in the outer nuclear layer (ONL), lengthening and disorganization in photoreceptor segments, and substantial reduction in cellularity and thickness of the inner nuclear layer (INL). The degenerative changes in the INL and ONL were most marked in the central and paracentral areas of the scrapie retinas, and were accompanied in all scrapie retinas by PrPSc deposition in the ganglion cell and synaptic layers. GFAP immunoreactivity was mainly increased in the ganglion cell and inner plexiform layers. Conclusions No appreciable fundoscopic changes were observed in the scrapie-affected ewes although reproducible changes in retinal function as measured by ERG were observed in these animals. The alterations in the receptoral and post-receptoral pathways corresponded to the degenerative lesions observed in the ONL and INL of the scrapie retinas. The retinal degeneration was associated with prion protein infectivity which presumably spread via the optic nerve.

Abstract Background Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant. Methods In this study we used the SELDI-TOF MS technology to analyze a large number of serum samples from control sheep and animals with early phase or late phase scrapie. A few potential low molecular weight biomarkers were selected by statistical methods and, after a training analysis, a protein signature pattern, which discriminates between early phase scrapie samples and control sera was identified. Results The combination of early phase biomarkers showed a sensitivity of 87% and specificity of 90% for all studied sheep in the early stage of the disease. One of these potential biomarkers was identified and validated in a SELDI-TOF MS kinetic study of sera from Syrian hamsters infected by scrapie, by western blot analysis and ELISA quantitation. Conclusions Differential protein expression profiling allows establishing a TSE diagnostic in scrapie sheep, in the early phase of the disease. Some proteic differences observed in scrapie sheep exist in infected hamsters. Further studies are being performed to identify all the discriminant biomarkers of interest and to test our potential markers in a new cohort of animals.

Mammalian prions are neurotropic pathogens formed from PrP Sc assemblies, a misfolded variant of the host-encoded prion protein PrP C . Multiple PrP Sc conformations or strains self-propagate in host populations or mouse models of prion diseases, exhibiting distinct biological and biochemical phenotypes. Constrained interactions between PrP Sc and PrP C conformations confer species specificity and regulate cross-species transmission. The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant PrP (rPrP) has been instrumental in demonstrating the protein-only nature of prions. Yet, their ability to encode different strains and transmit between species remains poorly studied, hampering their use in exploring structure-to-strain relationships. Fibrillar assemblies from rPrP with hamster, mouse, human, and bovine primary structures were generated and tested for transmission and adaptation in tg7 transgenic mice expressing hamster PrP C . All assemblies, except the bovine ones, were fully pathogenic on the primary passage, causing clinical disease, PrP Sc brain deposition, and spongiform degeneration. They exhibited divergent adaptation processes and strain properties upon subsequent passage. Assemblies of hamster origin propagated without apparent need for adaptation, those of mouse origin adapted abruptly, and those of human origin required serial passages for optimal fitness. Molecular analyses revealed the presence of endogenously truncated PrP Sc species in the resulting synthetic strains that lack the 90-140 amino acid region considered crucial for infectivity. In conclusion, rPrP assemblies provide a facile means of generating novel prion strains with adaptative/evolutive properties mimicking genuine prions. The PrP amino acid backbone is sufficient to encode different strains with specific adaptative properties, offering insights into prion transmission and strain diversity., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

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 (Δ 69-77 ). 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., (© 2024. The Author(s).)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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 PrP Sc immunohistochemistry staining in the brainstem and the absence of detectable PrP Sc 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., (© 2024. The Author(s).)

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 PrP Sc 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., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

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 PrP Sc , which is comprised of aggregated misfolded conformers of the normal host protein PrP C . 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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

To date, chronic wasting disease (CWD) is the most infectious form of prion disease affecting several captive, free ranging and wild cervid species. Responsible for marked population declines in North America, its geographical spread is now becoming a major concern in Europe. Polymorphisms in the prion protein gene (PRNP) are an important factor influencing the susceptibility to prions and their rate of propagation. All reported cervid PRNP genotypes are affected by CWD. However, in each species, some polymorphisms are associated with lower attack rates and slower progression of the disease. This has potential consequences in terms of genetic selection, CWD diffusion and strain evolution. CWD also presents a zoonotic risk due to prions capacity to cross species barriers. This review summarizes our current understanding of CWD control, focusing on PRNP genetic, strain diversity and capacity to infect other animal species, including humans., (© 2021. The Author(s).)

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrP Sc  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 PrP Sc  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., (© 2021. The Author(s).)

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 (M1 CJD and V2 CJD ). 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 V2 CJD strain or a mixture of M1 CJD  + V2 CJD 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., (© 2021. The Author(s).)

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 10 6 -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 10 5 -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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrP Sc in post-mortem tissues as indication of infection and disease. Since PrP Sc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrP Sc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrP Sc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Prions are neurotropic pathogens composed of misfolded assemblies of the host-encoded prion protein PrP C which replicate by recruitment and conversion of further PrP C by an autocatalytic seeding polymerization process. While it has long been shown that mouse-adapted prions cannot replicate and are rapidly cleared in transgenic PrP 0/0 mice invalidated for PrP C , these experiments have not been done with other prions, including from natural resources, and more sensitive methods to detect prion biological activity. Using transgenic mice expressing human PrP to bioassay prion infectivity and RT-QuIC cell-free assay to measure prion seeding activity, we report that prions responsible for the most prevalent form of sporadic Creutzfeldt-Jakob disease in human (MM1-sCJD) can persist indefinitely in the brain of intra-cerebrally inoculated PrP 0/0 mice. While low levels of seeding activity were measured by RT-QuIC in the brain of the challenged PrP 0/0 mice, the bio-indicator humanized mice succumbed at a high attack rate, suggesting relatively high levels of persistent infectivity. Remarkably, these humanized mice succumbed with delayed kinetics as compared to MM1-sCJD prions directly inoculated at low doses, including the limiting one. Yet, the disease that did occur in the humanized mice on primary and subsequent back-passage from PrP 0/0 mice shared the neuropathological and molecular characteristics of MM1-sCJD prions, suggesting no apparent strain evolution during lifelong dormancy in PrP 0/0 brain. Thus, MM1-sCJD prions can persist for the entire life in PrP 0/0 brain with potential disease potentiation on retrotransmission to susceptible hosts. These findings highlight the capacity of prions to persist and rejuvenate in non-replicative environments, interrogate on the type of prion assemblies at work and alert on the risk of indefinite prion persistence with PrP-lowering therapeutic strategies., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrP Sc , aberrant depositions of the host's cellular prion protein (PrP C ). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrP Sc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrP Sc . After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 10 6.2-6.4  ID 50 g -1 was reduced to a remaining infectivity of 10 4.6-5.7 ID 50 g -1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrP Sc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrP C . Leftover material in the remaining PrP Sc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

The diversity of goat scrapie strains in Europe has recently been studied using bioassays in a wide collection of rodent models, resulting in the classification of classical scrapie into four different categories. However, the sole use of the first passage does not lead to isolate adaptation and identification of the strains involved and might therefore lead to misclassification of some scrapie isolates. Therefore, this work reports the complete transmission study of a wide collection of goat transmissible spongiform encephalopathy (TSE) isolates by intracranial inoculation in two transgenic mouse lines overexpressing either small ruminant (TgGoat-ARQ) or bovine (TgBov) PrP C . To compare scrapie strains in sheep and goats, sheep scrapie isolates from different European countries were also included in the study. Once the species barrier phenomenon was overcome, an accurate classification of the isolates was attained. Thus, the use of just two rodent models allowed us to fully differentiate at least four different classical scrapie strains in small ruminants and to identify isolates containing mixtures of strains. This work reinforces the idea that classical scrapie in small ruminants is a prion disease caused by multiple different prion strains and not by a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE-C). In addition, the clear dissimilarity between the different scrapie strains and BSE-C does not support the idea that classical scrapie is the origin of epidemic BSE-C.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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 (PrP Sc ), 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 PrP C 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., (© 2020 Avar et al.)

Shadoo belongs to the prion protein family, an evolutionary conserved and extensively studied family due to the implication of PrP in Transmissible Spongiform Encephalopathies. However, the biological function of these genes remains poorly understood. While Sprn-knockdown experiments suggested an involvement of Shadoo during mouse embryonic development, Sprn-knockout experiments in 129Pas/C57BL/6J or 129Pas/FVB/NCr mice did not confirm it. In the present study, we analyzed the impact of Sprn gene invalidation in a pure FVB/NJ genetic background, using a zinc finger nuclease approach. The in-depth analysis of the derived knockout transgenic mice revealed a significant increase in embryonic lethality at early post-implantation stages, a growth retardation of young Sprn-knockout pups fed by wild type mice and a lactation defect of Sprn-knockout females. Histological and transcriptional analyses of knockout E7.5 embryos, E14.5 placentas and G7.5 mammary glands revealed specific roles of the Shadoo protein in mouse early embryogenesis, tissue development and differentiation with a potential antagonist action between PrP and Shadoo. This study thus highlights the entanglement between the proteins of the prion family.

An alternative method for the production of biodiesel from processed fats derived from Category 1, 2 and 3 animal by-products was assessed. The method is based on a pre-cleaning process, acidic esterification/transesterification of tallow using 1.5% methanesulfonic acid w/w; 140°C; 5.5 bar absolute pressure (bara); 4 h, followed by fractional distillation. The application focuses on the capacity of the alternative method to inactivate prions. Given the limitations that biodiesel presents for direct measurement of prion infectivity, the BIOHAZ Panel considered, based on the outcome of previous EFSA Opinions and current expert evaluation, that a reduction of 6 log 10 in detectable PrP S c signal would be necessary to consider the process at least equivalent to previously approved methods for Category 1 animal by-products. This is in addition to the inactivation achieved by the pressure sterilisation method applied before the application of any biodiesel production method. Experimental data were provided via ad hoc studies commissioned to quantify the reduction in detectable PrP S c in material spiked with scrapie hamster strain 263K, as measured by western blot, for the first two steps, with distillation assumed to provide at least an additional 3 log 10 reduction, based on published data. Despite the intrinsic methodological caveats of the detection of PrP S c in laboratory studies, the BIOHAZ Panel considers that the alternative method, including the final fractional distillation, is capable of achieving the required 6 log 10 reduction of the strain 263K PrP S c signal. Therefore, the method under assessment can be considered at least equivalent to the processing methods previously approved for the production of biodiesel from all categories of animal by-product raw materials. It is recommended to check the feasibility of the proposed HACCP plan by recording the main processing parameters for a certain time period under real industrial conditions., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Phenotypic variability in prion diseases, such as scrapie, is associated to the existence of prion strains, which are different pathogenic prion protein (PrP Sc ) conformations with distinct pathobiological properties. To faithfully study scrapie strain variability in natural sheep isolates, transgenic mice expressing sheep cellular prion protein (PrP C ) are used. In this study, we used two of such models to bioassay 20 scrapie isolates from the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, proteinase K-resistant PrP (PrP res ) banding patterns, lesion profiles and PrP Sc distribution were studied. Inocula showed a remarkable homogeneity on banding patterns, all of them but one showing 19-kDa PrP res . However, a number of isolates caused accumulation of 21-kDa PrP res in TgShp XI. A different subgroup of isolates caused long survival periods and presence of 21-kDa PrP res in Tg338 mice. It seemed that one major 19-kDa prion isoform and two distinct 21-kDa variants coexisted in source inocula, and that they could be separated by bioassay in each transgenic model. The reason why each model favours a specific component of the mixture is unknown, although PrP C expression level may play a role. Our results indicate that coinfection with more than one substrain is more frequent than infection with a single component.

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the "protein-only" hypothesis, the normal host-encoded prion protein (PrP C ) is converted into a pathological and infectious form (PrP Sc ) in these diseases. Transgenic knockout models have shown that PrP C is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrP C synthesis. We inoculated 12 goats (4 PRNP +/+ , 4 PRNP +/Ter , and 4 PRNP Ter/Ter ) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP +/+ goats and 773 dpi in PRNP +/Ter goats. PrP Sc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrP Sc was limited in peripheral organs, but all PRNP +/+ goats and 1 of 4 PRNP +/Ter goats were positive in head lymph nodes. The four PRNP Ter/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrP Sc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrP C due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrP C levels.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrP Sc ) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrP Sc (PrP res ) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrP res N-terminus. Suitable PrP res markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.

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- PRNP 129MM 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Transmissible spongiform encephalopathies (TSEs) or prion diseases of animals notably include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and classical bovine spongiform encephalopathy (C-BSE). As the transmission barrier phenomenon naturally limits the propagation of prions from one species to another, and the lack of epidemiological evidence for an association with human prion diseases, the zoonotic potential of these diseases was for a long time considered negligible. However, in 1996, C-BSE was recognized as the cause of a new human prion disease, variant Creutzfeldt-Jakob disease (vCJD), which triggered an unprecedented public health crisis in Europe. Large-scale epidemio-surveillance programs for scrapie and C-BSE that were implemented in the EU after the BSE crisis revealed that the distribution and prevalence of prion diseases in the ruminant population had previously been underestimated. They also led to the recognition of new forms of TSEs (named atypical) in cattle and small ruminants and to the recent identification of CWD in Europe. At this stage, the characterization of the strain diversity and zoonotic abilities associated with animal prion diseases remains largely incomplete. However, transmission experiments in nonhuman primates and transgenic mice expressing human PrP clearly indicate that classical scrapie, and certain forms of atypical BSE (L-BSE) or CWD may have the potential to infect humans. The remaining uncertainties about the origins and relationships between animal prion diseases emphasize the importance of the measures implemented to limit human exposure to these potentially zoonotic agents, and of continued surveillance for both animal and human prion diseases., (© 2019 International Society of Neuropathology.)