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.

Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrP. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrP. However, whether cofactors determine these different PrP conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrP to PrP. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.

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 (Bv109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrPSc (PrPres) in the same brain regions. Despite the low PrPres levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 108,4 i.c. ID50 infectious units per gram. Bv109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv109ICWD 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. Bv109ICWD showed unique characteristics of “virulence”, low PrPres accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity.

Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited neurodegenerative disorder associated with mutations in the prion protein gene and accumulation of misfolded PrP with protease-resistant fragments (PrPres) of 6–8 kDa. With the exception of a few GSS cases characterized by co-accumulation of PrPres of 21 kDa, efforts to transmit GSS to rodents have been unsuccessful. As a result, GSS subtypes exclusively associated with 6–8 kDa PrPres have often been considered as non-transmissible proteinopathies rather than true prion diseases. We show that GSS with P102L, A117V and F198S mutations transmit efficiently and produce distinct pathological phenotypes in bank voles (M. glareolus), irrespective of the presence of 21 kDa PrPres in the inoculum, demonstrating that GSS is a genuine prion disease characterized by both transmissibility and strain variation.

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.

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. Transmissible spongiform encephalopathies (TSEs) or prion diseases include fatal neurological diseases of humans and animals which can have different origin and epidemiology, i.e. acquired, sporadic or familial. Prion diseases are caused by a conformational switch of soluble cellular prion protein monomers (PrP C) into misfolded, partially protease-resistant and insoluble prion protein aggregates (PrP Sc), which then propagate through an autocatalytic conformational conversion.

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP Sc ). PrP Sc was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP Sc concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP Sc in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP Sc in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.