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The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant prion protein (rPrP) has been key to the demonstration that prions are infectious proteins responsible for human and animal transmissible spongiform encephalopathies. Yet, their use in identifying which structural PrP features are important for prion biology, including strain properties and capacity to transmit between species, has been hampered by their limited transmissibility de novo. We report the generation of prions with distinct biological characteristics from rPrP assemblies differing only in their primary structure (hamster, mouse and human amino acid sequence). These rPrP assemblies were transmissible to transgenic mice expressing hamster PrP, causing a clinical disease at full attack rate, brain deposition of pathological prion protein PrPScand spongiform degeneration. Their adaptation process on serial sub-passaging seemed to depend, as for genuine prions, on the presence of a species/transmission barrier, due notably to PrP sequence mismatch. Remarkably, one of the strains obtained is an unprecedented shortened prion, lacking the 90-140 amino-acid region which is believed to be key to infectivity and structural stability of disease-associated PrP assemblies. Finally, we provide evidence that rPrP prionogenicity lies in the structural organization and/or heterogeneity of the rPrP assemblies. These preparations of rPrP offer unprecedented opportunities for meaningful studies correlating the dynamicity and structures of PrPScassemblies to prion pathobiology.Author summaryPrions are infectious proteins, causing rapidly progressive neurodegenerative diseases in animals and humans. They are formed from the assisted-refolding and aggregation of the host-encoded prion protein (PrP). During the propagation of the disease, pathological PrP forces normal PrP to adopt its own conformation by a self-templating process. In infected host, different pathological structures of PrP or strains are found, causing diseases with specific biological phenotypes. Prions can also transmit between species. This capacity is limited by a species barrier, which critically depends on the infecting strain and PrP primary structure. How strain biological information is encoded in PrP structural fold remains unknown. We describe here the generation of differentbona fideprion strains with markedly distinct adaptation capacities by transmission of refolded assemblies derived from bacterially-derived recombinant PrP (rPrP) of different species. We provide evidence that pathogenicity lies in the structural organization and/or heterogeneity of rPrP assemblies. Pathological PrP from one of the generated strains exhibited unique molecular features, including absence of domains that are thought to be key to prion infectivity, according to most recent ultrastructural studies. Our findings provide new insights for generating prion infectious material and resolving mechanisms of infectivity acquisition during PrP conversion process.

Prion propagation can be interfered with by the expression of a second prion protein in the host. In the present study, we investigated prion propagation in a host expressing two different prion protein genes., Early studies in transgenic mouse lines have shown that the coexpression of endogenous murine prion protein (PrPC) and transgenic PrPC from another species either inhibits or allows the propagation of prions, depending on the infecting prion strain and interacting protein species. The way whereby this phenomenon, so-called “interference,” is modulated remains to be determined. In this study, different transgenic mouse lines were crossbred to produce mice coexpressing bovine and porcine PrPC, bovine and murine PrPC, or murine and porcine PrPC. These animals and their respective hemizygous controls were inoculated with several prion strains from different sources (cattle, mice, and pigs) to examine the effects of the simultaneous presence of PrPC from two different species. Our results indicate interference with the infection process, manifested as extended survival times and reduced attack rates. The interference with the infectious process was reduced or absent when the potentiality interfering PrPC species was efficiently converted by the inoculated agent. However, the propagation of the endogenous murine PrPSc was favored, allowing us to speculate that host-specific factors may disturb the interference caused by the coexpression of an exogenous second PrPC.

sCJD occurrence is currently assumed to result from spontaneous and stochastic formation of a misfolded PrP nucleus in the brains of affected patients. This original nucleus then recruits and converts nascent PrPC into PrPSc, leading to the propagation of prions in the patient’s brain. Our study demonstrates the coexistence of two prion strains in the brains of a majority of the 23 sCJD patients investigated. The relative proportion of these sCJD strains varied both between patients and between brain areas in a single patient. These findings strongly support the view that the replication of an sCJD prion strain in the brain of a patient can result in the propagation of different prion strain subpopulations. Beyond its conceptual importance for our understanding of prion strain properties and evolution, the sCJD strain mixture phenomenon and its frequency among patients have important implications for the development of therapeutic strategies for prion diseases., Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently classified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K-digested abnormal prion protein (PrPres) isoform identified by Western blotting (type 1 or type 2). Converging evidence led to the view that MM/MV1, VV/MV2, and VV1 and MM2 sCJD cases are caused by distinct prion strains. However, in a significant proportion of sCJD patients, both type 1 and type 2 PrPres were reported to accumulate in the brain, which raised questions about the diversity of sCJD prion strains and the coexistence of two prion strains in the same patient. In this study, a panel of sCJD brain isolates (n = 29) that displayed either a single or mixed type 1/type 2 PrPres were transmitted into human-PrP-expressing mice (tgHu). These bioassays demonstrated that two distinct prion strains (M1CJD and V2CJD) were associated with the development of sCJD in MM1/MV1 and VV2/MV2 patients. However, in about 35% of the investigated VV and MV cases, transmission results were consistent with the presence of both M1CJD and V2CJD strains, including in patients who displayed a “pure” type 1 or type 2 PrPres. The use of a highly sensitive prion in vitro amplification technique that specifically probes the V2CJD strain revealed the presence of the V2CJD prion in more than 80% of the investigated isolates, including isolates that propagated as a pure M1CJD strain in tgHu. These results demonstrate that at least two sCJD prion strains can be present in a single patient.

Prion infectivity was recently identified in the blood of both sporadic and variant Creutzfeldt-Jakob disease (CJD) patients. In variant CJD (vCJD), the widespread distribution of prions in peripheral tissues of both asymptomatic and symptomatic patients is likely to explain the occurrence of the observed prionaemia. However, in sporadic CJD (sCJD), prion infectivity is described to be located principally in the central nervous system. In this study, we investigated the presence of prion infectivity in bone marrow collected after death in patients affected with different sCJD agents. Bioassays in transgenic mice expressing the human prion protein revealed the presence of unexpectedly high levels of infectivity in the bone marrow from seven out of eight sCJD cases. These findings may explain the presence of blood-borne infectivity in sCJD patients. They also suggest that the distribution of prion infectivity in peripheral tissues in sCJD patients could be wider than currently believed, with potential implications for the iatrogenic transmission risk of this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

International audience; Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrPres accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrPres positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain.

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M 142 , N/D 146 , S/D 146 , R/Q 211 , and Q/K 222 ) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I 142 R 154 R 211 Q 222 ) goats and homozygous and/or heterozygous I/M 142 , R/H 154 , R/Q 211 , and Q/K 222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M 142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H 154 , Q 211 , and K 222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H 154 and Q 211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K 222 and a small proportion of homozygous K 222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K 222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.