A single amino acid residue in bank vole prion protein drives permissiveness to Nor98/atypical scrapie and the emergence of multiple strain variants.

Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein (PrPC) into infectious aggregates (PrPSc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrPSc. The transmissibility of prions depends on the PrPC sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.

Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America.

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.

Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease.

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrPSc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrPSc aggregates, whose protease-resistant core (PrPres) encompasses the whole C-terminus of PrP. In contrast, PrPSc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrPSc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (109.3 ID50 U/g) and is resistant to treatment with proteinase K (109.0 ID50 U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrPres in the different fractions, alongside the morphological characteristics of purified PrPres aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrPres, although the yield of PrPres was low. We found that this low yield depended on the low density/small size of GSS-A117V PrPres, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrPres, with infectivity levels that directly correlated with the PrPres amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrPresparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrPres, spanning residues ∼90-150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrPSc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.

Prion Disease in Dromedary Camels, Algeria.

Prions cause fatal and transmissible neurodegenerative diseases, including Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE). After the BSE epidemic, and the associated human infections, began in 1996 in the United Kingdom, general concerns have been raised about animal prions. We detected a prion disease in dromedary camels (Camelus dromedarius) in Algeria. Symptoms suggesting prion disease occurred in 3.1% of dromedaries brought for slaughter to the Ouargla abattoir in 2015-2016. We confirmed diagnosis by detecting pathognomonic neurodegeneration and disease-specific prion protein (PrPSc) in brain tissues from 3 symptomatic animals. Prion detection in lymphoid tissues is suggestive of the infectious nature of the disease. PrPSc biochemical characterization showed differences with BSE and scrapie. Our identification of this prion disease in a geographically widespread livestock species requires urgent enforcement of surveillance and assessment of the potential risks to human and animal health.

Strain-Dependent Morphology of Reactive Astrocytes in Human- and Animal-Vole-Adapted Prions.

Reactive astrogliosis is one of the pathological hallmarks of prion diseases. Recent studies highlighted the influence of several factors on the astrocyte phenotype in prion diseases, including the brain region involved, the genotype backgrounds of the host, and the prion strain. Elucidating the influence of prion strains on the astrocyte phenotype may provide crucial insights for developing therapeutic strategies. Here, we investigated the relationship between prion strains and astrocyte phenotype in six human- and animal-vole-adapted strains characterized by distinctive neuropathological features. In particular, we compared astrocyte morphology and astrocyte-associated PrPSc deposition among strains in the same brain region, the mediodorsal thalamic nucleus (MDTN). Astrogliosis was detected to some extent in the MDTN of all analyzed voles. However, we observed variability in the morphological appearance of astrocytes depending on the strain. Astrocytes displayed variability in thickness and length of cellular processes and cellular body size, suggesting strain-specific phenotypes of reactive astrocytes. Remarkably, four out of six strains displayed astrocyte-associated PrPSc deposition, which correlated with the size of astrocytes. Overall, these data show that the heterogeneous reactivity of astrocytes in prion diseases depends at least in part on the infecting prion strains and their specific interaction with astrocytes.

Effect of PrP genotype and route of inoculation on the ability of discriminatory Western blot to distinguish scrapie from sheep bovine spongiform encephalopathy.

Procedures for discriminating scrapie from bovine spongiform encephalopathy (BSE) in sheep are relevant to ascertain whether BSE has entered the sheep population. This study was aimed at investigating whether the suitability of an official EU discriminative method is affected by the sheep PrP genotype and route of infection.

Gerstmann-Sträussler-Scheinker disease subtypes efficiently transmit in bank voles as genuine prion diseases.

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 (PrP(res)) of 6-8 kDa. With the exception of a few GSS cases characterized by co-accumulation of PrP(res) of 21 kDa, efforts to transmit GSS to rodents have been unsuccessful. As a result, GSS subtypes exclusively associated with 6-8 kDa PrP(res) 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 PrP(res) in the inoculum, demonstrating that GSS is a genuine prion disease characterized by both transmissibility and strain variation.

Biochemical characterization of prion strains in bank voles.

Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrPSc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrPSc biochemical characteristics and if it was possible to univocally identify strains through PrPSc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived) and analyzed PrPSc from individual voles by epitope mapping of protease resistant core of PrPSc (PrPres) and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrPSc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed.

A new method for the characterization of strain-specific conformational stability of protease-sensitive and protease-resistant PrPSc.

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 PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) 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 PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) 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 PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) 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 PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

PrPSc in salivary glands of scrapie-affected sheep.

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.

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy.

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.

Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie.

The association between PrP gene variations and scrapie susceptibility was studied in a single herd of Ionica breed goats. The entire herd comprised 100 animals, 11 of which were clinically affected and showed pathological prion protein (PrP(Sc)) deposition in both their central nervous system (CNS) and lymphoreticular system (LRS). Among asymptomatic goats, nine harboured PrP(Sc) in both CNS and LRS, 19 showed PrP(Sc) only at the LRS level and 61 animals had no PrP(Sc) deposition. Genetic analysis of the PrP gene coding sequence revealed the presence of several polymorphisms, namely G37V, T110P, H143R, R154H, Q222K and P240S. Silent polymorphisms were also found at codons 42, 138, 219 and 232. The effect of PrP polymorphism on scrapie susceptibility was assessed by comparing the genotype distribution at each locus among animals with different pathogenetic and clinical disease stages. Significant differences in the distribution of genotypes were observed for codons 154 and 222, with polymorphism at codon 154 modulating susceptibility to scrapie and lysine at codon 222 being associated with scrapie resistance. The allelic variant encoding lysine at position 222 could be a valuable candidate to select in the framework of appropriate breeding programmes for scrapie resistance in goats.

Molecular analysis of cases of Italian sheep scrapie and comparison with cases of bovine spongiform encephalopathy (BSE) and experimental BSE in sheep.

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP(Sc) from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP(Sc). Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP(Sc) from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP(Sc). Scrapie cases displayed homogeneous PrP(Sc) features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP(Sc). While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.