An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

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.

Follow-up and monitoring programme in children identified in early-stage type 1 diabetes during screening in the general population of Italy.

AIM: To provide guidance for follow-up and monitoring of children and adolescents identified as positive to islet autoantibodies (IA) in the general population screening for type 1 diabetes (T1D) in Italy. METHODS: Detection of IA helps to diagnose pre-symptomatic T1D, prevent diabetic ketoacidosis (DKA) and identify persons for new therapies to delay symptomatic diabetes. Italy recently became the first country to approve by law a general autoantibody screening program for T1D and celiac disease in all children and adolescents (age 1-17yr). A pilot study is currently underway in four Italian regions addressing feasibility issues to be used in the scale up to nationwide screening. Meanwhile, a group of experts developed guidance recommendations for follow-up and monitoring of identified IA positive persons. RESULTS: Ten key components have been identified: establishment of a registry for children and adolescents at risk; close collaboration with the national network of family paediatricians; creation of T1D centers with expertise in follow-up and monitoring; educational measures; assurance of solid IA tests; identification of appropriate metabolic tests; feed-back feasibility and acceptability questionnaires; potential access to available therapeutic interventions; valuable outcome measures including DKA incidence; costs monitoring. Distinctive features of this program include single (in addition to multiple) IA antibody-positive persons in follow-up and the use of CGM to assess risk progression, rather than the cumbersome OGTT. CONCLUSION: It is expected that the proposed follow-up and monitoring program will be effective, affordable and acceptable to children and families identified in general T1D screening in Italy.

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.

Cofactor and glycosylation preferences for in vitro prion conversion are predominantly determined by strain conformation.

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrPC, into a pathogenic conformer, PrPSc. Infectious prions can exist as different strains, composed of unique conformations of PrPSc that generate strain-specific biological traits, including distinctive patterns of PrPSc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrPC glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrPC substrate or by the conformation of PrPSc seed. To distinguish between these two possibilities, we used bank vole PrPC to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di- or un-glycosylated bank vole PrPC substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrPC substrates when reactions contained the same PrPC glycoform or cofactor molecule preferred by the PrPSc seed in its host species. Thus, we conclude that it is the conformation of the input PrPSc seed, not the amino acid sequence of the PrPC substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrPC glycoforms during prion replication.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Full restoration of specific infectivity and strain properties from pure mammalian prion protein.

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrPSc, a misfolded conformer of the normal prion protein, PrPC. However, protein-only PrPSc 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 PrPSc in vitro. The restoration reaction is rapid, potent, and requires bank vole PrPC 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 PrPSc accurately stores latent infectious and strain information, but cofactor molecules control a reversible switch that unmasks biological infectivity.

Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

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 replication without host adaptation during interspecies transmissions.

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.

Variable Protease-Sensitive Prionopathy Transmission to Bank Voles.

Variably protease-sensitive prionopathy (VPSPr), a recently described human sporadic prion disease, features a protease-resistant, disease-related prion protein (resPrPD) displaying 5 fragments reminiscent of Gerstmann-Sträussler-Scheinker disease. Experimental VPSPr transmission to human PrP-expressing transgenic mice, although replication of the VPSPr resPrPD profile succeeded, has been incomplete because of second passage failure. We bioassayed VPSPr in bank voles, which are susceptible to human prion strains. Transmission was complete; first-passage attack rates were 5%-35%, and second-passage rates reached 100% and survival times were 50% shorter. We observed 3 distinct phenotypes and resPrPD profiles; 2 imitated sporadic Creutzfeldt-Jakob disease resPrPD, and 1 resembled Gerstmann-Sträussler-Scheinker disease resPrPD. The first 2 phenotypes may be related to the presence of minor PrPD components in VPSPr. Full VPSPr transmission confirms permissiveness of bank voles to human prions and suggests that bank vole PrP may efficiently reveal an underrepresented native strain but does not replicate the complex VPSPr PrPD profile.

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

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.

Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway.

Chronic wasting disease (CWD) persists in cervid populations of North America and in 2016 was detected for the first time in Europe in a wild reindeer in Norway. We report the detection of CWD in 3 moose (Alces alces) in Norway, identified through a large scale surveillance program. The cases occurred in 13-14-year-old female moose, and we detected an abnormal form of prion protein (PrPSc) in the brain but not in lymphoid tissues. Immunohistochemistry revealed that the moose shared the same neuropathologic phenotype, characterized by mostly intraneuronal deposition of PrPSc. This pattern differed from that observed in reindeer and has not been previously reported in CWD-infected cervids. Moreover, Western blot revealed a PrPSc type distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. These findings suggest that these cases in moose represent a novel type of CWD.

Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease.

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrPTSE) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrPTSE MVAG), showing that PrPTSE MVAG is composed of multiple conformers with biochemical properties distinct from those of PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns, and PrPTSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MVAGIMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Isolation of a Defective Prion Mutant from Natural Scrapie.

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155-231 and ∼80-231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of "authentic" defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90-155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

Cofactors influence the biological properties of infectious recombinant prions.

Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc 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 PrPC to PrPSc. 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.

Chronic wasting disease in bank voles: characterisation of the shortest incubation time model for prion diseases.

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(109I)CWD), typified by unprecedented short incubation times of 25-28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv(109I)CWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv(109I)CWD 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(109I)CWD 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(109I)CWD 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(109I)CWD 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.

L-type bovine spongiform encephalopathy in genetically susceptible and resistant sheep: changes in prion strain or phenotypic plasticity of the disease-associated prion protein?

BACKGROUND: Sheep with prion protein (PrP) gene polymorphisms QQ171 and RQ171 were shown to be susceptible to the prion causing L-type bovine spongiform encephalopathy (L-BSE), although RQ171 sheep specifically propagated a distinctive prion molecular phenotype in their brains, characterized by a high molecular mass protease-resistant PrP fragment (HMM PrPres), distinct from L-BSE in QQ171 sheep. METHODS: The resulting infectious and biological properties of QQ171 and RQ171 ovine L-BSE prions were investigated in transgenic mice expressing either bovine or ovine PrP. RESULTS: In both mouse lines, ovine L-BSE transmitted similarly to cattle-derived L-BSE, with respect to survival periods, histopathology, and biochemical features of PrPres in the brain, as well as splenotropism, clearly differing from ovine classic BSE or from scrapie strain CH1641. Nevertheless and unexpectedly, HMM PrPres was found in the spleen of ovine PrP transgenic mice infected with L-BSE from RQ171 sheep at first passage, reminiscent, in lymphoid tissues only, of the distinct PrPres features found in RQ171 sheep brains. CONCLUSIONS: The L-BSE agent differs from both ovine classic BSE or CH1641 scrapie maintaining its specific strain properties after passage in sheep, although striking PrPres molecular changes could be found in RQ171 sheep and in the spleen of ovine PrP transgenic mice.

Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.