In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv^109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv^109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv^109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrP^Sc (PrP^res) in the same brain regions. Despite the low PrP^res levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 10^8,4 i.c. ID₅₀ infectious units per gram. Bv^109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv^109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv^109ICWD showed unique characteristics of “virulence”, low PrP^res accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrP^Sc, neurodegeneration and infectivity. Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids and is expanding increasingly in the USA and Canada. Animal models are of key importance in the study of prion diseases but their development for CWD has long been hampered by its very inefficient transmission to wild-type mice. Significant progress was made following the generation of transgenic mice over-expressing cervid PrP. Here we show that the bank vole (Myodes glareolus), a wild rodent species that we demonstrated to be susceptible to many animal and human prion diseases, is also very susceptible to CWD from elk, mule deer and white-tailed deer. Adaptation of CWD to bank vole led to the isolation of a prion strain with peculiar characteristics: unprecedented short incubation and survival times, respectively of 25–28 and ∼35 days, low PrP^Sc levels compared with other vole-adapted prion strains and high infectious titre. These features were all faithfully maintained upon the generation of this strain in vitro by protein misfolding cyclic amplification. The development of a model for prion diseases that led to disease in less than one month accumulating high infectious titres but low PrP^Sc levels, represents a significant tool for investigating the still unclear relationship between PrP^Sc, neurodegeneration and infectivity in prion diseases.

Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe. Significance Chronic wasting disease (CWD) is a highly contagious disease caused by prions that affects several cervid species and is relentlessly spreading across North America. Very recently, CWD was detected for the first time in Europe. In this study, we found that Norwegian CWD strains are distinct from those causing the epidemic in North America. Moreover, we show that Norwegian reindeer and moose are affected by different CWD strains, revealing an unprecedented prion strain variation in Norwegian wild cervid populations. These findings indicate that North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases and have implications for CWD control strategies in Europe, as well as for the safety of humans.

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrP^Sc), which is derived from its cellular precursor (PrP^C), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrP^C and Sho, we inferred that PrP^C levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrP^C glycosylation and proteolytic processing, net reductions in PrP^C occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrP^C results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrP^Sc in vitro. While PrP^C downregulation is not discernible in animals with unusually short incubation periods and high PrP^C expression, slowly evolving prion infections exhibit downregulation of the PrP^C substrate required for new PrP^Sc synthesis and as a receptor for pathogenic signaling. Our data reveal PrP^C downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP^Sc, a misfolded conformer of the normal prion protein, PrP^C. However, protein-only PrP^Sc preparations lack significant levels of prion infectivity, leading to the alternative hypothesis that cofactor molecules are required to form infectious prions. Here, we show that prions with parental strain properties and full specific infectivity can be restored from protein-only PrP^Sc in vitro. The restoration reaction is rapid, potent, and requires bank vole PrP^C substrate, post-translational modifications, and cofactor molecules. To our knowledge, this represents the first report in which the essential properties of an infectious mammalian prion have been restored from pure PrP without adaptation. These findings provide evidence for a unified hypothesis of prion infectivity in which the global structure of protein-only PrP^Sc accurately stores latent infectious and strain information, but cofactor molecules control a reversible switch that unmasks biological infectivity. Prions are unusual infectious agents that cause invariably fatal brain diseases. Unlike conventional infectious agents such as bacteria or viruses, prions do not possess nucleic acids such as DNA or RNA, and therefore it is not clear how they are able to replicate and cause infection. A leading model is that prions are composed exclusively of a specific protein molecule with an abnormal shape, which has the ability to coerce other protein molecules to change into the same abnormal shape in a self-reinforcing process. Although this model is attractive, no one has ever been able to make potently infectious prions from only pure protein. Here, we show for the first time that pure protein can faithfully store and transmit specific infectious information (strain properties) in a latent state even though it is non-infectious.

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

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions. Prion disorders are a group of devastating neurodegenerative diseases caused by an aberrantly folded isoform of the endogenous prion protein. The molecular mechanisms by which this proteinaceous pathogen is able to propagate in the central nervous system and cause neuronal death are poorly understood, partially due to the difficulties elucidating the three-dimensional structure of the aggregation-prone aberrant isoform or prion. Obtaining sufficient amounts of highly pure and homogeneous prions for high-resolution structural studies has been limited until now due to technical reasons. Here, we present a novel method for the production of large amounts of highly infectious recombinant prions suitable for solid state Nuclear Magnetic Resonance imaging, which could help to unveil the molecular pathogenesis of these particular pathogens.