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Prion diseases are neurodegenerative disorders caused by a change in conformation of the prion protein from the cellular form (PrP C ) to a misfolded isoform (PrP Sc ). PrP C is a copper binding protein via histidine residues in the octapeptide repeats (OR) and the non-OR region located at the N-terminus. Although the functional implication of copper binding to PrP C is still under investigation, copper may play a role in prion disease. In this study, we describe transgenic mice expressing mouse prion protein replacing histidine 95 by tyrosine (PrP H95Y) to disrupt the non-OR copper-binding site. Transgenic mice overexpressing PrP H95Y showed clinical signs and died at about 100 days with spongiform degeneration and PK-resistant PrP. Inoculation of brain homogenate from mice overexpressing PrP H95Y to Tga20 mice expressing wild-type PrP also causes lethal, spongiform encephalopathy. We conclude that this substitution could promote PrP C -PrP Sc conversion and induce spontaneous prion disease in vivo .

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.

Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate.

Prion diseases are caused by the conversion of physiological PrP C into the pathogenic misfolded protein PrP Sc , conferring new properties to PrP Sc that vary upon prion strains. In this work, we analyze the thermostability of three prion strains (BSE, RML and 22L) that were heated at 98 °C for 2 hours. PrP Sc resistance to proteinase K (PrP res ), residual infectivity by mouse bioassay and in vitro templating activity by protein misfolding cyclic amplification (PMCA) were studied. Heated strains showed a huge loss of PrP res and a radically different infectivity loss: RML was the most thermolabile strain (6 to 7 log10 infectivity loss), followed by 22L (5 log10) while BSE was the most thermostable strain with low or null infectivity reduction showing a clear dissociation between PrP res and infectivity. These results indicate that thermostability is a strain-specific feature, measurable by PMCA and mouse bioassay, and a great tool to distinguish prion strains.

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.

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.

Classical bovine spongiform encephalopathy (BSE) is the only zoonotic prion disease described to date. Although the zoonotic potential of atypical BSE prions have been partially studied, an extensive analysis is still needed. We conducted a systematic study by inoculating atypical BSE isolates from different countries in Europe into transgenic mice overexpressing human prion protein (PrP): TgMet(129), TgMet/Val(129), and TgVal(129). L-type BSE showed a higher zoonotic potential in TgMet(129) mice than classical BSE. whereas Val(1)(29)-PrP variant was a strong molecular protector against L-type BSE prions, even in heterozygosis. H-type BSE could not be transmitted to any of the mice. We also adapted 1 H- and 1 L-type BSE isolate to sheep-PrP transgenic mice and inoculated them into human-PrP transgenic mice. Atypical BSE prions showed a modification in their zoonotic ability after adaptation to sheep-PrP producing agents able to infect TgMet(129) and TgVal(129) bearing features that make them indistinguishable of sporadic Creutzfeldt-Jakob disease prions.

E/D 163 polymorphism of dog prion protein (PrP) has been recently proposed as the variant responsible for canid prion resistance. To further investigate the protective role of this variant against prion replication, the transgenic mouse model OvPrP-Tg532 expressing sheep/goat PrP carrying the substitution D 162 (equivalent to D 163 position of dog PrP) was generated and intracranially inoculated with a broad collection of small ruminant prion strains. OvPrP-Tg532 mice showed resistance to classical bovine spongiform encephalopathy (BSE) from sheep and some classical scrapie isolates from sheep and goat but were susceptible to ovine atypical L-BSE and numerous classical scrapie isolates. Strikingly, some of these classical scrapie isolates showed a shift in their prion strain properties. These results suggest that other PrP residues apart from E/D 163 variant of dog PrP or factors distinct than PrP may participate in prion resistance of canids and that different factors may be required for D 162 sheep PrP to provide effective protection to sheep against ruminant prions.

Background More than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date. Methods We describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro PrP Sc propagation studies were performed using recombinant-adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease. Results The index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated PrP Sc fragment, consistent with a Gerstmann-Sträussler-Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of PrP Sc studies failed to show disease transmissibility. Conclusion In conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be “probably damaging”, heterozygous carriers are protected, at least from an early onset providing evidence for a potentially recessive pattern of inheritance in human prion diseases.

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA). In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology. All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate. Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious. This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer's Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).