Distribution and quantitative estimates of variant Creutzfeldt Jakob Diseases prions in the 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

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.info:eu-repo/semantics/acceptedVersio

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.This work was funded by FEDER POCTEFA TRANSPRION (EFA282/13) and REDPRION (EFA148/16), by the UK Food Standards Agency Exploring permeability of the species barrier (M03043 and FS231051), by the European Union through FP7 222887 “Priority”, the Spanish Ministerio de Economía y Competitividad [AGL2016-78054-R (AEI/FEDER, UE). A.M.-M. was supported by a fellowship from the INIA (FPI-SGIT-2015-02), and P.A.-C. was supported by a fellowship from the Spanish Ministerio de Economía y Competitividad (BES-2010-040922)

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

17 Pág.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 PrPC IMPORTANCE 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.This work was funded by EU Projects FOOD-CT-2006-36353 (Goat-BSE) and CT2009-222887 (Priority) and by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) and a fellowship (BES-2010-040922) to P.A.-C. A.M.-M. was supported by a fellowship from the INIA (FPI-SGIT-2015-02).Peer reviewe

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

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

A Simple, Versatile and Sensitive Cell-Based Assay for Prions from Various Species

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies

Évaluation du risque de transmission iatrogène des maladies à prions

Les Encéphalopathies Spongiformes Transmissibles (EST ou maladies à Prions) sont des maladies neurodégénératives fatales caractérisées par l'accumulation d'un conformère anormal (PrPSc) d'une protéine de l'hôte (PrP). Chez chacune des espèces affectées, les prions présentent une diversité biologique (souches) à laquelle sont associées des caractéristiques phénotypiques et des capacités à se propager à des espèces hétérologues différentes. De manière assez surprenante, les propriétés biologiques des souches de prion semblent capables d'évoluer spontanément ou au gré du franchissement de barrières d'espèces (transmission entre un donneur et un receveur d'espèces hétérologues). Dans une perspective de santé publique, les risques sanitaires associés aux agents des ESTs peuvent être rangés dans deux catégories (i) Le franchissement de barrière de transmission ; c'est-à-dire la capacité potentielle ou avérée d'une souche prion circulant chez une espèce donnée à se propager (dans des conditions naturelles ou accidentelles d'exposition) à un hôte appartenant à une espèce hétérologue. L'Encéphalopathie Spongiforme Bovine (ESB) constitue l'exemple le plus emblématique de ce risque ; l'exposition alimentaire de l'Homme à des bovins contaminés par l'ESB étant responsable de l'émergence du variant de la maladie de Creutzfeldt-Jakob. (ii) Le risque de transmission iatrogène ; c'est-à-dire la transmission accidentelle d'une maladie à Prions consécutive à une procédure médicale. Chez l'Homme, plusieurs centaines de cas de transmission iatrogène de MCJ ont été identifiés. Les risques de transmission iatrogène semblent dépendre de la nature des souches de prion et de la distribution de l'infectiosité dans l'organisme des patients atteints ou en incubation de la maladie (tissus périphériques et fluides corporels). Dans ce travail, nous avons caractérisé la perméabilité de diverses 'barrières d'espèces' d'intérêt (potentiel zoonotique ou transmission à des espèces d'animaux de rente) relatives aux souches de prion responsables de formes atypiques de l'ESB (ESB-L et ESB-H) ou de tremblante (Nor98). Nous avons mis en évidence une plasticité, jusqu'alors insoupçonnée, des propriétés biologiques de ces agents et documenté leur capacité à générer, au gré de leur propagation, des prions capables de franchir la barrière d'espèce humaine. En parallèle, nous nous sommes intéressés à la diversité des souches de prion responsables des formes sporadiques ou iatrogènes de MCJ chez l'Homme avant d'en caractériser la distribution (et les quantités d'infectiosité) dans les tissus périphériques de patients atteints de vMCJ et sMCJ. Les résultats obtenus contredisent l'idée selon laquelle, les prions responsables de sMCJ restent limités au système nerveux central ; une hypothèse qui a conduit les évaluateurs de risques à considérer que les fluides biologiques (sang, urine) et les tissus périphériques humains présentaient un risque faible ou négligeable de transmettre cette maladie. Nos résultats contribuent à documenter les risques sanitaires associés aux prions animaux et humains. Ils sont susceptibles d'aider les agences de santé à adapter les mesures actuellement déployées pour prévenir les risques de transmission des ESTs animales et humaines.Transmissible Spongiform Encephalopathies (TSEs or Prion diseases) are fatal neurodegenerative diseases characterised by the accumulation of an abnormal isoform (PrPSc) of a host encoded protein (PrP). In affected species, prions display biological diversity (strains) that are associated with specific phenotypic characteristics and abilities to transmit to different heterologous species. Surprisingly, these strain properties can evolve spontaneously or after transmission through species barriers (transmission from a donor to different species recipient). From a public health perspective, the risks associated with TSE agents fall into two categories: (i) Transmission barrier passages, i.e. the potential or proven capacity of a prion strain to transmit (under natural or accidental exposure conditions) to hosts belonging to a heterologous species. Bovine Spongiform Encephalopathy (BSE) constitutes the most emblematic example for this risk; human dietary exposure to BSE-contaminated cattle is responsible for the emergence of variant Creutzfeldt-Jakob disease. (ii) The iatrogenic transmission risk, i.e. the accidental transmission of a Prion disease via a medical procedure. In humans, several hundred of iatrogenic CJD cases have been identified. Iatrogenic transmission risks are apparently dependent on the nature of prion strains and on the infectivity distribution in the organism of affected/incubating patients (peripheral tissues and body fluids). In this work, we characterised the permeability of various 'species barriers' of interest (zoonotic potential or transmission to livestock species) associated with prion strains that are responsible for atypical forms of BSE (BSE-L and BSE-H) or scrapie (Nor98). We highlighted a previously unsuspected plasticity of the biological properties of these agents and documented their capacity to generate, as they replicate, prions able to cross the human species barrier. In parallel, we investigated the diversity of prion strains responsible for sporadic or iatrogenic forms of CJD before characterising their distribution (and infectivity quantities) in the peripheral tissues of vCJD and sCJD affected patients. The obtained results contradict the view that in affected patients sCJD prions remain limited to CNS; an assumption that led risk assessors to consider that biological fluids (blood, urine) and human peripheral tissues present a low or negligible risk of transmitting this disease. Our results contribute to documenting the sanitary risks associated with animal and human prions. They could help the public health agencies to adapt the measures currently implemented to prevent the transmission risk associated to animal and human TSE agents

Prion diseases iatrogenic transmission risks

Les Encéphalopathies Spongiformes Transmissibles (EST ou maladies à Prions) sont des maladies neurodégénératives fatales caractérisées par l'accumulation d'un conformère anormal (PrPSc) d'une protéine de l'hôte (PrP). Chez chacune des espèces affectées, les prions présentent une diversité biologique (souches) à laquelle sont associées des caractéristiques phénotypiques et des capacités à se propager à des espèces hétérologues différentes. De manière assez surprenante, les propriétés biologiques des souches de prion semblent capables d'évoluer spontanément ou au gré du franchissement de barrières d'espèces (transmission entre un donneur et un receveur d'espèces hétérologues). Dans une perspective de santé publique, les risques sanitaires associés aux agents des ESTs peuvent être rangés dans deux catégories (i) Le franchissement de barrière de transmission ; c'est-à-dire la capacité potentielle ou avérée d'une souche prion circulant chez une espèce donnée à se propager (dans des conditions naturelles ou accidentelles d'exposition) à un hôte appartenant à une espèce hétérologue. L'Encéphalopathie Spongiforme Bovine (ESB) constitue l'exemple le plus emblématique de ce risque ; l'exposition alimentaire de l'Homme à des bovins contaminés par l'ESB étant responsable de l'émergence du variant de la maladie de Creutzfeldt-Jakob. (ii) Le risque de transmission iatrogène ; c'est-à-dire la transmission accidentelle d'une maladie à Prions consécutive à une procédure médicale. Chez l'Homme, plusieurs centaines de cas de transmission iatrogène de MCJ ont été identifiés. Les risques de transmission iatrogène semblent dépendre de la nature des souches de prion et de la distribution de l'infectiosité dans l'organisme des patients atteints ou en incubation de la maladie (tissus périphériques et fluides corporels). Dans ce travail, nous avons caractérisé la perméabilité de diverses 'barrières d'espèces' d'intérêt (potentiel zoonotique ou transmission à des espèces d'animaux de rente) relatives aux souches de prion responsables de formes atypiques de l'ESB (ESB-L et ESB-H) ou de tremblante (Nor98). Nous avons mis en évidence une plasticité, jusqu'alors insoupçonnée, des propriétés biologiques de ces agents et documenté leur capacité à générer, au gré de leur propagation, des prions capables de franchir la barrière d'espèce humaine. En parallèle, nous nous sommes intéressés à la diversité des souches de prion responsables des formes sporadiques ou iatrogènes de MCJ chez l'Homme avant d'en caractériser la distribution (et les quantités d'infectiosité) dans les tissus périphériques de patients atteints de vMCJ et sMCJ. Les résultats obtenus contredisent l'idée selon laquelle, les prions responsables de sMCJ restent limités au système nerveux central ; une hypothèse qui a conduit les évaluateurs de risques à considérer que les fluides biologiques (sang, urine) et les tissus périphériques humains présentaient un risque faible ou négligeable de transmettre cette maladie. Nos résultats contribuent à documenter les risques sanitaires associés aux prions animaux et humains. Ils sont susceptibles d'aider les agences de santé à adapter les mesures actuellement déployées pour prévenir les risques de transmission des ESTs animales et humaines.Transmissible Spongiform Encephalopathies (TSEs or Prion diseases) are fatal neurodegenerative diseases characterised by the accumulation of an abnormal isoform (PrPSc) of a host encoded protein (PrP). In affected species, prions display biological diversity (strains) that are associated with specific phenotypic characteristics and abilities to transmit to different heterologous species. Surprisingly, these strain properties can evolve spontaneously or after transmission through species barriers (transmission from a donor to different species recipient). From a public health perspective, the risks associated with TSE agents fall into two categories: (i) Transmission barrier passages, i.e. the potential or proven capacity of a prion strain to transmit (under natural or accidental exposure conditions) to hosts belonging to a heterologous species. Bovine Spongiform Encephalopathy (BSE) constitutes the most emblematic example for this risk; human dietary exposure to BSE-contaminated cattle is responsible for the emergence of variant Creutzfeldt-Jakob disease. (ii) The iatrogenic transmission risk, i.e. the accidental transmission of a Prion disease via a medical procedure. In humans, several hundred of iatrogenic CJD cases have been identified. Iatrogenic transmission risks are apparently dependent on the nature of prion strains and on the infectivity distribution in the organism of affected/incubating patients (peripheral tissues and body fluids). In this work, we characterised the permeability of various 'species barriers' of interest (zoonotic potential or transmission to livestock species) associated with prion strains that are responsible for atypical forms of BSE (BSE-L and BSE-H) or scrapie (Nor98). We highlighted a previously unsuspected plasticity of the biological properties of these agents and documented their capacity to generate, as they replicate, prions able to cross the human species barrier. In parallel, we investigated the diversity of prion strains responsible for sporadic or iatrogenic forms of CJD before characterising their distribution (and infectivity quantities) in the peripheral tissues of vCJD and sCJD affected patients. The obtained results contradict the view that in affected patients sCJD prions remain limited to CNS; an assumption that led risk assessors to consider that biological fluids (blood, urine) and human peripheral tissues present a low or negligible risk of transmitting this disease. Our results contribute to documenting the sanitary risks associated with animal and human prions. They could help the public health agencies to adapt the measures currently implemented to prevent the transmission risk associated to animal and human TSE agents

Prion strains are differentially released through the exosomal pathway

International audienceCell-to-cell transfer of prions is a crucial step in the spreading of prion infection through infected tissue. At the cellular level, several distinct pathways including direct cell-cell contacts and release of various types of infectious extracellular vesicles have been described that may potentially lead to infection of na < ve cells. The relative contribution of these pathways and whether they may vary depending on the prion strain and/or on the infected cell type are not yet known. In this study we used a single cell type (RK13) infected with three different prion strains. We showed that in each case, most of the extracellular prions resulted from active cell secretion through the exosomal pathway. Further, quantitative analysis of secreted infectivity indicated that the proportion of prions eventually secreted was dramatically dependent on the prion strain. Our data also highlight that infectious exosomes secreted from cultured cells might represent a biologically pertinent material for spiking experiments. Also discussed is the appealing possibility that abnormal PrP from different prion strains may differentially interact with the cellular machinery to promote secretion