Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions
- Jérôme Chapuis†1,
- Mohammed Moudjou†1,
- Fabienne Reine†1,
- Laetitia Herzog†1,
- Emilie Jaumain1,
- Céline Chapuis1,
- Isabelle Quadrio2, 3,
- Jacques Boulliat4,
- Armand Perret-Liaudet2, 3,
- Michel Dron1,
- Hubert Laude1,
- Human Rezaei1 and
- Vincent Béringue1Email author
© Chapuis et al. 2016
Received: 7 January 2016
Accepted: 26 January 2016
Published: 5 February 2016
Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals. These diseases can occur spontaneously, such as Creutzfeldt-Jakob disease (CJD) in humans, or be acquired or inherited. Prions are primarily formed of macromolecular assemblies of the disease-associated prion protein PrPSc, a misfolded isoform of the host-encoded prion protein PrPC. Within defined host-species, prions can exist as conformational variants or strains. Based on both the M/V polymorphism at codon 129 of PrP and the electrophoretic signature of PrPSc in the brain, sporadic CJD is classified in different subtypes, which may encode different strains. A transmission barrier, the mechanism of which remains unknown, limits prion cross-species propagation. To adapt to the new host, prions have the capacity to ‘mutate’ conformationally, leading to the emergence of a variant with new biological properties. Here, we transmitted experimentally one rare subtype of human CJD, designated cortical MM2 (129 MM with type 2 PrPSc), to transgenic mice overexpressing either human or the VRQ allele of ovine PrPC.
In marked contrast with the reported absence of transmission to knock-in mice expressing physiological levels of human PrP, this subtype transmitted faithfully to mice overexpressing human PrP, and exhibited unique strain features. Onto the ovine PrP sequence, the cortical MM2 subtype abruptly evolved on second passage, thereby allowing emergence of a pair of strain variants with distinct PrPSc biochemical characteristics and differing tropism for the central and lymphoid tissues. These two strain components exhibited remarkably distinct replicative properties in cell-free amplification assay, allowing the ‘physical’ cloning of the minor, lymphotropic component, and subsequent isolation in ovine PrP mice and RK13 cells.
Here, we provide in-depth assessment of the transmissibility and evolution of one rare subtype of sporadic CJD upon homologous and heterologous transmission. The notion that the environment or matrix where replication is occurring is key to the selection and preferential amplification of prion substrain components raises new questions on the determinants of prion replication within and between species. These data also further interrogate on the interplay between animal and human prions.
Mammalian prions are proteinaceous pathogens responsible for a broad range of fatal neurodegenerative diseases in humans and animals . Prions are primarily formed of macromolecular assemblies of PrPSc, a misfolded, ß-sheet enriched form of the ubiquitously expressed, plasma membrane-anchored, variably N-glycosylated and α-helix rich, host-encoded prion protein PrPC . This change is based on the self-sustained transfer of a structural information from the PrPSc conformer in the prion state to PrPC, presumably through a seeding-polymerization process . Within defined host species, PrPC can transconform in multiple prion variants or strains, differing in their PrPSc conformations at the level of the tertiary and/or quaternary structure, in their biological properties and in their relative capacity to replicate in cell lines or tissues [5, 8, 14, 21, 37]. Prions can propagate within and between species, as exemplified by the emergence of human variant Creutzfeldt-Jakob disease (vCJD) through dietary exposure to prions responsible for the bovine spongiform encephalopathy (BSE) epidemics in cattle . Within defined species, prions can also form sporadically. In humans, the incidence of sporadic Creutzfeldt-Jakob disease (sCJD) ranges between 1 and 2 cases per million and per year, and affects mainly elderly people . Atypical BSEs (L-type and H-type) and atypical scrapie Nor98 are thought to develop spontaneously in aged ruminants .
Characterizing PrPSc electrophoretic pattern following limited digestion with proteinase K (PK) can allow differentiation of prion strains. For sCJD, methionine/valine (M/V) polymorphism at codon 129 of the gene encoding PrP and the migration pattern and relative glycoform abundance of PK-resistant PrPSc (PrPres) allow the definition of different molecular subtypes [28, 45, 57]. These subtypes exhibit specific clinical and neuropathological features [28, 45]. The most common form of sCJD is associated with the presence of type 1 (T1) PrPres and homozygosity for methionine at codon 129. The unglycosylated band of T1 PrPres migrates at 21 kDa in SDS-PAGE gels, and monoglycosylated forms predominate over diglycosylated ones. Rare forms of MM sporadic CJD with a Type 2 (T2) PrPres type, - the unglycosylated form of which migrating at 19 kDa -, have been diagnosed. These forms are further subclassified as cortical and thalamic variants [40, 45, 51]. The cortical variant is distinguished from all other sporadic forms by the absence of experimental transmission to knock-in mouse models expressing human PrP at physiological levels [10, 30, 40].
Mice transgenic for PrP have been instrumental in deciphering prion strain diversity and in modeling experimentally the so-called species or transmission barrier that limits prions interspecies transmission (for review ). In essence, such mice are generated to express specific sequences from mammalian PrPC on a mouse PrP-ablated background, and are inoculated with prions. Analyzing the clinical outcome, attack rate and the presence of PrPSc in brain and peripheral tissues where prion replication can occur [5, 27] allows the establishment of whether cross-interactions between host PrPC and invading PrPSc structural landscapes are possible with regard to prion conversion. The conformational hypothesis [21, 60] posits that PrPC can adopt a limited portfolio of conformations in the PrPSc state, due to structural constraints in its amino acid backbone. If the infecting PrPSc conformation(s) is within the portfolio of possible conformations, cross-species transmission will occur. If not, the transmission barrier will be high, and can lead to an abrupt change in prion strain biological properties [2, 4, 5, 9, 13, 29, 38, 46, 47, 54], a phenomenon referred to as a ‘mutation’. Whether the newly emerging strain is selected from an ensemble of pre-existing PrPSc conformations in the original inoculum (‘quasi-species’) or is generated ex abrupto remains difficult to determine. A high transmission barrier does not lead systematically to prion strain ‘mutation’, as highlighted by the remarkable ability of classical BSE prions to retain their biological properties, despite intermediate passage to a range of different hosts [12, 15, 35].
Here, we studied the strain biological properties of one rare subtype of sporadic CJD prions, the cortical MM2-form, upon transmission to either human or ovine PrP transgenic mice.
Materials and methods
All animal experiments were approved by the Local Ethics Committee of the authors’ institution (Comethea; permit number 12/034). Human tissues samples were selected from the tissue bank of the French National Neuropathology Network for CJD on the basis of the availability of autopsy-retained frozen brain material and informed consent from patient’s relatives for autopsy and research use, according to the French regulation (L.1232-1 to L.1232-3, Code de la Santé Publique).
Transgenic mouse lines
The human PrP tg650 and ovine PrP tg338 lines have been described previously [5, 7, 34]. These lines are homozygous with approximately 6-fold and 8-fold over-expression of human PrPC (M129 allele) and ovine PrPC (V136R154Q171 allele) in brain, respectively.
MM2-sCJD transmission and titration
To avoid any cross-contamination, a strict protocol based on the use of disposable equipment and preparation of all inocula in a class II microbiological cabinet was followed. A cerebellum extract was used as source of sporadic CJD, cortical MM2 subtype. This sample has a T2 specific PrPres molecular profile. The tissue extract was prepared as 10 % w/v homogenate in 5 % w/v glucose with a Precellys (Ozyme, Montigny-le-Bretonneux, France) for inoculation into tg650 and tg338 mice. Twenty microliters were inoculated intracerebrally in the right hemisphere to groups of individually identified 6-8 week-old tg650 or tg338 mice, at the level of the parietal cortex. The first mouse succumbing with disease was used for subpassaging. The brain and spleen (when specified) of this mouse were collected with distinct, disposable tools, homogenized at 20 % w/v in 5 % glucose and reinoculated intracerebrally at 10 % w/v.
For endpoint titration, starting from 10 % w/v brain homogenate (‘undiluted’ material), serial 10-fold dilutions of brain homogenates were prepared in 5 % w/v glucose containing 5 % w/v bovine serum albumin. Twenty microliters of each dilution were immediately inoculated into individually identified 6-8-week-old tg650 or tg338 recipient mice by intracerebral route. Animals were supervised daily for the appearance of neurological signs associated with the development of a prion disease. Animals at terminal stage of disease or at end of life were euthanized. The brains and spleens of all animals were analyzed for PrPres content. The number of prion-positive mice was used to establish, by the Spearman-Kärber method, the number of prion infectious units per gram of tissue leading to median mouse infection (ID50 per gram).
Protein misfolding cyclic amplification (PMCA)
Mouse brain lysates from healthy tg338 mice were used as the substrate for tg650-passaged MM2-sCJD and tg338-adapted MM2-sCJD prion seeds. One round of PMCA, except indicated otherwise, was performed as previously described , in a microplate format, on a Q700 sonicator (Delta Labo, Colombelles, France), consisting in 96 cycles of 30 s of sonication at 200- to 220-W power followed by 29 min 30 s of incubation at 37 °C.
RK13 cells and the Rov P2FJ6 clone expressing constitutively the VRQ allele of ovine PrP (tg2 construct; ) were routinely cultured in Opti-MEM, derived of the Eagle's minimal essential medium (opti-MEM; Gibco), supplemented with 10% fetal calf serum and antibiotics (penicillin, streptomycin), as previously described . Cells were cultured at 37°C in a humidified 5 % CO2 atmosphere in a cell culture incubator. Cell monolayers in 12-well plate were exposed to brain or spleen homogenates from tg338 mice infected with ovine MM2-sCJD prions diluted in opti-MEM. Cells were exposed to 20 μl of 20 % brain homogenate or 40 μl of 10 % spleen homogenate diluted in 1 ml of fresh medium. One week later, cells were transferred to one 25-cm2 flask for growing. Each week (i.e. one passage), the cells were then split at the ¼ dilution in two flasks, one for further cultivation, one for PrPres content analysis, as previously described . Dissociation of the cells was made with a cell-dissociating buffer (Sigma).
PrPres was extracted from 20 % (wt/vol.) tissue homogenates with the Bio-Rad TeSeE detection kit, as previously described . Briefly, aliquots were digested with proteinase K (200 μg/ml final concentration) for 10 min at 37 °C before B buffer precipitation and centrifugation at 28,000 × g for 15 min. Pellets were resuspended in sample buffer, denatured, run on 12 % Bis/Tris gels (Bio-Rad), electrotransferred onto nitrocellulose membranes, and probed with 0.1 μg/ml biotinylated anti-PrP monoclonal antibody Sha31 antibody (human PrP epitope 145-152, ) or with 0.1 μg/ml anti-PrP 12B2 antibody (human PrP epitope 89-93, epitope, ) and followed by streptavidin conjugated to horseradish peroxidase (HRP) or by HRP conjugated to goat anti-mouse IgG1 antibody (1/20 000 final dilution), respectively . Immunoreactivity was visualized by chemiluminescence (GE Healthcare). The size and relative amounts of PrPres glycoforms were determined by the use of GeneTools software after acquisition of chemiluminescent signals with a GeneGnome digital imager (Syngene, Frederick, MD).
Enzymatic deglycosylation was performed on denatured PrPres with 1,000 U of recombinant PNGase (New England BioLabs, Evry, France) for 2h at 37 °C in 1 % Nonidet P40 and the proprietary buffer. The deglycosylated proteins were then precipitated with three volumes of cold acetone and resuspended in Laemmli sample buffer for western blot analysis as described above.
Brains were rapidly removed from euthanized mice and frozen on dry ice. Cryosections were cut at 8-10 μm, transferred onto Superfrost slides and kept at -20 °C until use. Histoblot analyses were performed as described , using the 12F10 anti-PrP antibody (human PrP epitope 142-160, ). Analysis was performed with a digital camera (Coolsnap, Photometrics) mounted on a binocular glass (SZX12, Olympus). The sections presented are representative of the analysis of three brains samples.
Description of the MM2-sCJD case
Faithful propagation and unique strain properties of MM2-cortical sCJD prions in human PrP mice
Serial transmission of MM2-cortical sCJD to mice expressing human or ovine PrP
Mean incubation timeb (n/n0)
tg650 (human PrP)
tg338 (ovine PrP)
278 ± 6 (6/6)
557 ± 11 (6/7)
274 ± 4 (8/8)
84 ± 1 (6/6)
280 ± 4 (12/12)
81 ± 2 (6/6)
268 ± 4 (8/8)a
78 ± 2 (7/7)
80 ± 1 (6/6)
After each passage, the nervous and lymphoid tissues of the diseased mice were examined for the presence of PrPres, by immunoblotting. PrPres was readily detected in the brains of all the mice analyzed. A typical T2 banding pattern, characterized by aglycosyl PrPres migrating at 19 kDa and prominent monoglycoform species was observed (Fig. 1a-b), as in the human MM2-cortical case. This signature was conserved on subsequent passage and differed from that observed after transmission of the other sCJD subtypes to tg650 mice, as shown for comparison. Those elicited accumulation of PrPres with a T1 signature alone (MM1) or concomitant with the presence of low-size fragments in variable amounts (MV1, VV1, VV2, MV2, Fig. 1a-d; Additional file 1: Figure S1). Only serial transmission of L-type BSE prions to tg650 mice produced a T2 signature in the brain (shown as T2 control in Fig. 1c, [4, 6]).
A number of PrPres typing studies revealed the co-existence of T1 and T2 PrPres in the brain of a variable percentage of sCJD cases [16, 48, 53, 57]. Analysis of PrPres pattern in the brain of MM2-sCJD inoculated tg650 mice, either by using the 12B2 anti-PrP antibody that specifically recognizes T1 PrPres or after deglycosylation failed to detect the T1 isoform (Fig. 1c-d).
Assessment of the neuroanatomical distribution of PrPres by histoblotting revealed that MM2-sCJD prions deposited in the dorsal and habenular thalamic nuclei, in the optic tract, in the cingulum, in the external capsule, in the lateral hypothalamic area and in the trigeminal nuclei of tg650 brain (Fig. 1e).
Endpoint titration of serially passaged MM2-sCJD prions in human or ovine PrP mice
Mean incubation timea (n/n0)
tg650 (human PrP)
tg338 (ovine PrP)
351 ± 6 (5/5)
441 ± 51 (3/5)
163 ± 10 (6/6)
424; 501 (2/6)
227 ± 22 (4/6)
> 500 (0/6)
Collectively, these results indicate that cortical MM2-sCJD prions propagated with no transmission barrier in human PrP tg650 mice. The biological strain phenotype observed in these mice was unique amongst the other sCJD or vCJD sources transmitted so far.
Isolation of 2 prion strain types with preferential tissue tropism on serial transmission of MM2-sCJD to ovine PrP mice
The same human source of MM2-sCJD was serially transmitted by intracerebral route to mice expressing the VRQ allele of ovine PrP (tg338 line; ), so as to assess both the cross-species transmission capacity of MM2-sCJD prions and the convertibility of ovine PrPVRQ by foreign prions. On primary passage, six out of the seven inoculated mice developed a neurologic disease, with a mean incubation time of 557 ± 11 days, suggesting that propagation onto the ovine PrP sequence has occurred readily. On second passage, the mean incubation was dramatically shortened to approximately 80 days, a value that remained stable over 4 subpassages (Table 1). This abrupt change in the incubation time between the 1st and the 2nd passage is consistent with the isolation of a shorter incubation period variant [2, 4, 5, 8, 9, 13, 29, 46, 47, 54].
Spleens from diseased mice gradually accumulate detectable levels of PrPres over subpassaging (0/5 at 1st passage; 1/2 on 2nd passage, 5/5 on 3rd and subsequent passages). The spleen-banding pattern was homogeneous and distinct from that observed in the brain, the unglysosylated PrPres core migrating at ~21 kDa (Fig. 2a-b). This pattern was denominated T1Ov.
Transmission of MM2-sCJD prions to ovine PrP mice after PMCA amplification or passage through P2FJ6 cells
Mean incubation timea (n/n0)
80 ± 1 (6/6)
79 ± 1 (6/6)
MM2-sCJD → PMCA
105 ± 1 (5/5)
MM2-sCJD → PMCA → tg338 brain
84 ± 2 (6/6)
MM2-sCJD → PMCA → tg338 spleen
93 ± 2 (6/6)
MM2-sCJD → P2FJ6 cells
80 ± 1 (6/6)
Cloned MM2-sCJD → P2FJ6 cells
75 ± 2 (6/6)
MM2-sCJD → PMCA → P2FJ6 cells
118 ± 1 (6/6)
MM2-sCJD → RK13 cells → tg338
> 400 d (0/6)
Collectively, these results demonstrate that the serial transmission of MM2-sCJD prions to tg338 mice led, after an abrupt shortening of the incubation time, to the isolation of two strain components, a dominant T2Ov component in the brain and a T1Ov subcomponent that preferentially populates the spleen. Biological cloning of brain material allowed elimination of the T1Ov component, at least to levels below detectable replication in tg338 mice.
Preferential selection of the T1Ov substrain component by protein misfolding cyclic amplification
To ascertain whether the PMCA process results in the selective amplification of T1Ov prions, tg338 mice were intracerebrally challenged with products generated from the 10−8 brain seed, that is 100-fold below the lowest dose resulting in positive transmission by bioassay (Table 2). Tg338 mice succumbed with disease in 105 ± 1 days, a survival time that established to approximately 90 days on further subpassaging (Table 3). A unique T1Ov type electrophoretic signature was observed in both brains and spleens over these two passages, whether subpassage was performed with brain or spleen material (Fig. 4c, Table 3). After inoculation of brain or spleen material, PrPres deposition in the brain also differed from that observed with non amplified MM2-sCJD prions. PrPres deposited more ventrally in the lateral hypothalamic areas and granular-like deposits were observed in the corpus callosum (Fig. 3 c-d). This material was designated PMCA MM2-sCJD prions. Brains infected with PMCA MM2-sCJD prions were also submitted to PMCA reaction. The seeding activity detection limit established at the 10−9 dilution, as observed with uncloned MM2-sCJD prions passaged in tg338 mice (Fig. 4d).
The use of tg338-cloned MM2-sCJD brain material further demonstrated that T2Ov seeds were poor convertors in PMCA reaction. The limiting dilution achieved was dramatically decreased by 104-fold as compared to uncloned MM2-sCJD prions (Fig. 4d). PrPres banding pattern observed was still T1Ov, suggesting that one biological cloning was not sufficient to eliminate totally this subcomponent for PMCA reaction, owing to the improved sensitivity of this technique .
Collectively, these data indicated that the T1Ov subtype was an isolable substrain component of MM2-sCJD in tg338 mice, which carried most if not all the PMCA activity. Separately, the T1Ov subcomponent was pathogenic in tg338 mice, as T2Ov.
PMCA-generation of the T1Ov substrain component by seeding ovine PrPC with human MM2-sCJD prions
PMCA has been shown as a convenient model to investigate experimentally prion species barrier [3, 18, 25]. We thus examined the outcome of confronting directly human MM2-sCJD prions to ovine PrPC. Brain homogenates from uninfected tg338 mice were seeded with a 10−3 dilution of brain homogenate from tg650 mice infected with uncloned or cloned MM2-sCJD prions. To augment the chance of crossing the species barrier, up to three rounds of PMCA were performed. At each round, the amplified products were analyzed for PrPres content by Western blot. As shown in Fig. 4e (representative of n = 4 independent experiments), PrPres was detected at each round. The electrophoretic pattern observed resembled T1Ov, the unglysosylated PrPres core migrating at ~21 kDa. A T2Ov PrPres signature was occasionally observed in the PMCA products (1 out of 16 positive reactions, data not shown), suggesting possible stochastic generation of these prions on the ovine PrP sequence. Remarkably, the same efficacy of amplification relative to the number of rounds was found with either cloned or uncloned tg650 material, allowing detection of T1Ov PrPres from 105-fold diluted seeds in three rounds (Fig. 4e). It can be extrapolated from these data that under the constraint of having to be amplified by PMCA with an ovine PrP substrate, T1Ov prions can directly emerge from a human MM2-sCJD prions population, cloned or not.
The two MM2-sCJD ovine subcomponents can be stably passaged in a Rov cell subclone
RK13 cells expressing the VRQ allele of ovine PrP are permissive to ‘fast’ scrapie prions, serially passaged or not in tg338 mice [32, 56, 58]. Motivated by the short incubation times of MM2-sCJD prions in tg338 mice, we examined whether they could propagate in these cells and whether one subcomponent would compete out the other. The Rov9 cell clone  and congeners were poorly permissive to tg338-passaged MM2-sCJD prions. Cells mostly failed to accumulate detectable levels of PrPres, despite exposure to lowly diluted brain homogenate, and prolonged incubation of the cells with the inoculum (data not shown and Munoz-Montesino et al., submitted for publication). Another Rov construct constitutively expressing ovine PrP (P2FJ6 cells) and used routinely in our scrapie cell assays because of its marked susceptibility to scrapie 127S prions ([32, 56] and to be published elsewhere) was permissive to MM2-sCJD prions. To sustain replication, it was necessary to adapt the infection protocol, so as to allow longer time of exposure between the cells and the inoculum (see methods).
All tg338 mice inoculated with P2FJ6 cell lysates exposed to tg338 brain-passaged MM2-sCJD prions (at passage 7, to exclude remnant inoculum, as confirmed with RK13 cells) developed disease with a strain phenotype similar to MM2-sCJD prions in tg338 mice: the mean incubation time established at 80 dpi (Table 3); brain PrPres exhibited a T2Ov banding pattern (Fig. 5d), and neuroanatomical distribution of PrPres closely resembled that observed upon direct MM2-sCJD subpassaging (Fig. 3e). Immunoblot examination of the spleens revealed the presence of the T1Ov component (Fig. 5d), suggesting ‘silent’ propagation in the P2FJ6 cells.
To further ascertain that P2FJ6 cells could propagate T1Ov as T2Ov prions and exclude any in-cell reversal phenomenon between the two components, P2FJ6 cells were exposed to the two T2Ov and T1Ov subcomponents separately, by using brain material derived from tg338-cloned MM2-sCJD prions (i.e. enriched in T2Ov) or from PMCA MM2-sCJD prions (i.e. enriched in T1Ov), respectively. In that case, cells faithfully accumulated prions with T2Ov and T1Ov PrPres signatures, respectively (Fig. 5a). When cells were challenged with spleen material from the same mice, infection proceeded at slower rate, as shown by the lower levels of PrPres accumulation, which necessitated highly exposed gels to be visible (Fig. 5c). The PrPres signature observed were T2Ov from cloned material and T1Ov from PMCA passaged material (Fig. 5c), as in the spleen. Whatever the tissue used to infect the cells, the T1Ov and T2Ov signatures were kept up to 12 cell passages, suggesting stable cell replication of the two subcomponents independently (data not shown). Reinoculation of the so-called T2Ov and T1Ov cell lysates to tg338 mice confirmed the faithful and separate propagation of the two agents in the cells. Indeed their strain specific signature was preserved after intermediate passage in cells, with regard to incubation time (Table 3), PrPres banding pattern in brain and spleen tissues (Fig. 5d) and histoblot analyses (Fig. 3f-g). Altogether, these data indicate that Rov P2FJ6 cells were fully competent to replicate the two tg338 passaged MM2-sCJD infectious components.
We report here on the capacity of a rare subtype of human sCJD prions (MM2, cortical variant) to adapt onto the ovine PrP sequence, thereby modifying its apparent substrain composition and tissue tropism. We further show that the isolated variants can exhibit distinct replicative or biological behavior depending on the matrix or environment surrounding the prion conversion process.
Human PrP tg650 mice challenged with the MM2-cortical CJD subtype developed a clinical disease at full attack rate in less than 300 days. A T2 PrPres profile was invariably observed in the inoculated mice. These characteristics were maintained over passaging, suggesting faithful propagation of this agent in this mouse line. Together, the incubation time in tg650 mice and the brain PrPres profiles were unique among the panel of CJD cases transmitted so far to the tg650 line (Fig. 1 and unpublished data), thus fully supporting the contention that MM2-cortical CJD is a specific CJD subtype, as previously concluded from the absence of transmission to transgenic lines expressing physiological level of human PrP [10, 30, 40]. Human PrP overexpression in tg650 mice may have been key in the success of transmission. Additionally, we used a cerebellum extract as inoculum, whereas previously, a cerebral cortex extract was used. Different brains and different brain regions harbor prion infectivity titers that can vary by 100-fold with regard to the number of infectious units per milligram of tissue [1, 11]. While such a difference would barely affect the transmission rate of MM2-sCJD prions to tg650 mice (Table 2), this might be an issue in knock-in human PrP transgenic mice in which disease develops at slower pace .
While MM2-sCJD prions propagated faithfully in human PrP mice, the outcome was different upon transmission to ovine PrP tg338 mice. Efficient transmission occurred on primary passage, at near full attack rate, albeit with a prolonged incubation period. A drastic reduction in the incubation time from 550 to 80 days occurred on secondary passage, consistent with the isolation of a variant, or a ‘mutant’ by analogy to mutational events observable with conventional microorganisms [2, 4, 5, 8, 9, 13, 29, 46, 47, 54]. While a 19K-like PrPres signature predominated in tg338 mouse brains as in human or human PrP mouse brain, a second signature, designated T1Ov gradually emerged with subpassaging in tg338 mouse spleens. This distinct tissue tropism and the possibility to isolate, by conventional biological cloning or cell-free amplification, and further propagate separately and with fidelity the T2Ov and T1Ov types in tg338 mice or P2FJ6 cells indicate that a pair of bona fide prion strains has been isolated on the ovine PrP sequence.
Once propagated alone, we failed to evidence any gradual shift of T2OV towards T1Ov prions in tg338 mice or in P2FJ6 cells, suggesting that the interspecies prion conversion events have been instrumental in the emergence of the T1Ov substrain component. A recurrent and often perplexing question [8, 21, 60, 61] is whether these agents have been generated de novo on confrontation to the new PrP sequence or have been preferentially selected from pre-existing PrPSc conformations. Our data support the contention that T1Ov prions have been generated de novo: First, if the T1Ov agent preexisted in the human MM2-sCJD source, it was not able to replicate at detectable levels in the spleens of tg650 mice, at variance with other human prion strain sources [5, 6]. Second, we found no evidence of a T1 signature in the brain of tg650 mice inoculated with MM2-sCJD prions that may putatively be at the origin of T1Ov prions onto the ovine PrP sequence (Fig. 1). Third, T1Ov prions could be directly generated by PMCA from the human MM2-SCJD prions population passaged in tg650 mice. The lack of obvious difference in the PMCA efficacy between uncloned and cloned material (Fig. 4e), the latter being theoretically less populated in a potential T1Ov subcomponent (to be compared with the dramatic reduction in the PMCA efficacy when tg338-passaged uncloned and cloned MM2-sCJD prions are amplified, Fig. 4d), suggests de novo generation of T1Ov on confrontation to the ovine PrP sequence. We acknowledge that definitive proof is lacking and it is possible that substrain heterogeneity may constantly arise even in cloned populations during propagation .
Our observations are adding to the view that 2 distinct prion strains can propagate in neural and extraneural tissues of the same host individual [5, 7]. We are currently comparing the retro-transmission properties of the T1Ov and T2Ov types to human PrP mice to examine whether the selection pressure imposed by the heterologous transmission was lower in spleen than brain tissues from tg338 mice, as previously observed . The situation may however be drastically different here, as the T1Ov component emerged gradually in the spleen with subpassaging and adaptation, whereas previously, the spleen component emerged first, and at higher rate, than the brain component .
Endpoint titration of isolated T1Ov and T2Ov MM2-sCJD prions in ovine PrP mice
Mean incubation timea (n/n0)
142 ± 8 (5/5)
148 ± 4 (6/6)
174; 192 (2/6)c
212 ± 6 (3/6)c
> 450 (0/6)c
> 450 (0/6)c
> 450 (0/6)c
Within the quasi-species concept applied to prions, -which proposes that prions are not constituting a single clone but are embedded with all PrPSc conformational variants [21, 61]-, our observations would suggest that there has been a bottleneck event in tg338 mice (due to the heterologous PrP transmission) which has affected MM2-sCJD prions fitness, at least on primary passage, and has led to the emergence of two strain components. Key to the quasi-species definition is the existence of intra-population interactions, either complementation or interference . Complementation is difficult to accommodate with the observation that there is so far no apparent regeneration of the two T1Ov and T2Ov subcomponents after intermediate separation of one of the component, either by PMCA or biological cloning. As individuals, both components do not harbor different fitness with regard to incubation time, cell replicability and infectious titer; however, jointly, T2Ov markedly outcompetes T1Ov in either P2FJ6 cells or in tg338 brain. Neuroanatomically, the absence of replication of T1Ov prions in certain brain target areas, that are free of T2Ov prions (such as the lateral hypothalamic area (Fig. 3)) suggest local interfering mechanisms, independent of competition for the same PrP substrate . We thus propose that the MM2-sCJD ‘mutant’ isolated in tg338 mice is not a mere agglomeration of independently acting T1Ov and T2Ov conformations.
Since the discovery that RK13 cells expressing the ovine PrP VRQ allele were permissive to certain scrapie prions [23, 58], we and others have made numerous attempts to infect these cells or more permissive clones such as the P2FJ6 one with ovine prions sources, passaged or not onto tg338 mice. Most attempts have failed, except for prions classified as ‘fast’, as based on their short incubation time in reporter tg338 mice (e.g. 127S and LA21K fast, [42, 56]). Here we show that two other relatively ‘fast’ agents originating from MM2-sCJD can fairly propagate in Rov cells at their maximum infectivity levels, as assessed by the tg338 bioassay. Many factors can account for the difficulties to replicate prions in immortalized cell lines. Obviously, prion-doubling time must surpass the cell division rate. For ‘fast’ strains such as 127S and LA21K fast, it appears that a subset of small oligomers are by far the most active with respect to prion replication in vivo and to seeding activity by PMCA . This might be key to their sustained ability to replicate in cells. We are currently investigating the oligomeric state of the most active sCJD aggregates. In any case, this extended panel of strains replicating in cell culture opens the possibility to compare their biology in cells expressing wild type or mutated forms of PrP (Munoz-Montesino et al., submitted for publication).
We report here that prions associated with a cortical subtype of human MM2-sCJD prions converts the ovine VRQ PrP in its disease-associated form. The same source propagates efficiently in mice expressing the ARQ allele of ovine PrP (). Our ongoing studies indicate that human MM1 and MV2 CJD prions can also adapt on the VRQ allele of ovine PrP. This indicates that CJD prions exhibit a retro-zoonotic potential. Reversely, certain scrapie prions could propagate on human PrP mice  and in non-human primates which exhibit similar amino acid sequence as humans . Thus the human to ovine prion transmission barrier may not be absolute. From an evolutionary point of view, these data interrogate on the potential interplay between animal and human prions and their true origin, either spontaneous/sporadic versus infectious. Their fate may not be not as compartmentalized as commonly believed.
We thank the staff of Animalerie Rongeurs (INRA, Jouy-en-Josas, France) for animal care, Annick Le Dur and Tina Knaple for excellent technical help, Christelle Langevin (INRA, Jouy-en-Josas) for the generation of the P2F plasmid, and Jan Langeveld for the 12B2 anti-PrP monoclonal antibody. This work was partially funded by the Alliance Biosecure Foundation (BoHuToHu project), by the Ile de France Region (DIM MALINF) and by the Fondation pour la Recherche Médicale (Equipe FRM DEQ20150331689).
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- Andreoletti O, Orge L, Benestad SL, Beringue V, Litaise C, Simon S, et al. Atypical/Nor98 scrapie infectivity in sheep peripheral tissues. PLoS pathogens. 2011;7, e1001285. doi:10.1371/journal.ppat.1001285.PubMed CentralView ArticlePubMedGoogle Scholar
- Angers RC, Kang HE, Napier D, Browning S, Seward T, Mathiason C, et al. Prion strain mutation determined by prion protein conformational compatibility and primary structure. Science. 2010;328:1154–8. doi:10.1126/science.1187107.PubMed CentralView ArticlePubMedGoogle Scholar
- Barria MA, Telling GC, Gambetti P, Mastrianni JA, Soto C. Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions. The Journal of biological chemistry. 2011;286:7490–5. doi:10.1074/jbc.M110.198465.PubMed CentralView ArticlePubMedGoogle Scholar
- Beringue V, Andreoletti O, Le Dur A, Essalmani R, Vilotte JL, Lacroux C, et al. A bovine prion acquires an epidemic bovine spongiform encephalopathy strain-like phenotype on interspecies transmission. J Neurosci. 2007;27:6965–71. doi:10.1523/JNEUROSCI.0693-07.2007.View ArticlePubMedGoogle Scholar
- Beringue V, Herzog L, Jaumain E, Reine F, Sibille P, Le Dur A, et al. Facilitated cross-species transmission of prions in extraneural tissue. Science. 2012;335:472–5. doi:10.1126/science.1215659.View ArticlePubMedGoogle Scholar
- Beringue V, Herzog L, Reine F, Le Dur A, Casalone C, Vilotte JL, et al. Transmission of atypical bovine prions to mice transgenic for human prion protein. Emerging infectious diseases. 2008;14:1898–901. doi:10.3201/eid1412.080941.PubMed CentralView ArticlePubMedGoogle Scholar
- Beringue V, Le Dur A, Tixador P, Reine F, Lepourry L, Perret-Liaudet A, et al. Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD. PloS one. 2008;3, e1419. doi:10.1371/journal.pone.0001419.PubMed CentralView ArticlePubMedGoogle Scholar
- Beringue V, Vilotte JL, Laude H. Prion agent diversity and species barrier. Veterinary research. 2008;39:47. doi:10.1051/vetres:2008024.View ArticlePubMedGoogle Scholar
- Bessen RA, Marsh RF. Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. Journal of virology. 1992;66:2096–101.PubMed CentralPubMedGoogle Scholar
- Bishop MT, Will RG, Manson JC. Defining sporadic Creutzfeldt-Jakob disease strains and their transmission properties. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:12005–10. doi:10.1073/pnas.1004688107.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown P, Gibbs Jr CJ, Rodgers-Johnson P, Asher DM, Sulima MP, Bacote A, et al. Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease. Annals of neurology. 1994;35:513–29. doi:10.1002/ana.410350504.View ArticlePubMedGoogle Scholar
- Bruce M, Chree A, McConnell I, Foster J, Pearson G, Fraser H. Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philos Trans R Soc Lond B Biol Sci. 1994;343:405–11. doi:10.1098/rstb.1994.0036.View ArticlePubMedGoogle Scholar
- Bruce ME. Scrapie strain variation and mutation. Br Med Bull. 1993;49:822–38.PubMedGoogle Scholar
- Bruce ME. TSE strain variation. Br Med Bull. 2003;66:99–108.View ArticlePubMedGoogle Scholar
- Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature. 1997;389:498–501. doi:10.1038/39057.View ArticlePubMedGoogle Scholar
- Cali I, Castellani R, Alshekhlee A, Cohen Y, Blevins J, Yuan J, et al. Co-existence of scrapie prion protein types 1 and 2 in sporadic Creutzfeldt-Jakob disease: its effect on the phenotype and prion-type characteristics. Brain. 2009;132:2643–58. doi:10.1093/brain/awp196.PubMed CentralView ArticlePubMedGoogle Scholar
- Cassard H, Torres JM, Lacroux C, Douet JY, Benestad SL, Lantier F, et al. Evidence for zoonotic potential of ovine scrapie prions. Nature communications. 2014;5:5821. doi:10.1038/ncomms6821.View ArticlePubMedGoogle Scholar
- Castilla J, Gonzalez-Romero D, Saa P, Morales R, De Castro J, Soto C. Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions. Cell. 2008;134:757–68. doi:10.1016/j.cell.2008.07.030.PubMed CentralView ArticlePubMedGoogle Scholar
- Caughey B, Baron GS, Chesebro B, Jeffrey M. Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions. Annu Rev Biochem. 2009;78:177–204. doi:10.1146/annurev.biochem.78.082907.145410.PubMed CentralView ArticlePubMedGoogle Scholar
- Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci. 2001;24:519–50. doi:10.1146/annurev.neuro.24.1.519.View ArticlePubMedGoogle Scholar
- Collinge J, Clarke AR. A general model of prion strains and their pathogenicity. Science. 2007;318:930–6. doi:10.1126/science.1138718.View ArticlePubMedGoogle Scholar
- Comoy EE, Mikol J, Luccantoni-Freire S, Correia E, Lescoutra-Etchegaray N, Durand V, et al. Transmission of scrapie prions to primate after an extended silent incubation period. Sci Rep. 2015;5:11573. doi:10.1038/srep11573.PubMed CentralView ArticlePubMedGoogle Scholar
- Courageot MP, Daude N, Nonno R, Paquet S, Di Bari MA, Le Dur A, et al. A cell line infectible by prion strains from different species. J Gen Virol. 2008;89:341–7. doi:10.1099/vir.0.83344-0.View ArticlePubMedGoogle Scholar
- Feraudet C, Morel N, Simon S, Volland H, Frobert Y, Creminon C, et al. Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J Biol Chem. 2005;280:11247–58. doi:10.1074/jbc.M407006200.View ArticlePubMedGoogle Scholar
- Green KM, Castilla J, Seward TS, Napier DL, Jewell JE, Soto C, et al. Accelerated high fidelity prion amplification within and across prion species barriers. PLoS pathogens. 2008;4, e1000139. doi:10.1371/journal.ppat.1000139.PubMed CentralView ArticlePubMedGoogle Scholar
- Halliez S, Reine F, Herzog L, Jaumain E, Haik S, Rezaei H, et al. Accelerated, spleen-based titration of variant Creutzfeldt-Jakob disease infectivity in transgenic mice expressing human prion protein with sensitivity comparable to that of survival time bioassay. Journal of virology. 2014;88:8678–86. doi:10.1128/JVI.01118-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J. Species-barrier-independent prion replication in apparently resistant species. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:10248–53.PubMed CentralView ArticlePubMedGoogle Scholar
- Hill AF, Joiner S, Wadsworth JD, Sidle KC, Bell JE, Budka H, et al. Molecular classification of sporadic Creutzfeldt-Jakob disease. Brain. 2003;126:1333–46.View ArticlePubMedGoogle Scholar
- Kimberlin RH, Walker CA, Fraser H. The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. J Gen Virol. 1989;70(Pt 8):2017–25.View ArticlePubMedGoogle Scholar
- Kobayashi A, Iwasaki Y, Otsuka H, Yamada M, Yoshida M, Matsuura Y, et al. Deciphering the pathogenesis of sporadic Creutzfeldt-Jakob disease with codon 129 M/V and type 2 abnormal prion protein. Acta Neuropathol Commun. 2013;1:74. doi:10.1186/2051-5960-1-74.PubMed CentralView ArticlePubMedGoogle Scholar
- Krasemann S, Groschup MH, Harmeyer S, Hunsmann G, Bodemer W. Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice. Mol Med. 1996;2:725–34.PubMed CentralPubMedGoogle Scholar
- Laferriere F, Tixador P, Moudjou M, Chapuis J, Sibille P, Herzog L, et al. Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics. PLoS Pathog. 2013;9, e1003702. doi:10.1371/journal.ppat.1003702.PubMed CentralView ArticlePubMedGoogle Scholar
- Langeveld JP, Jacobs JG, Erkens JH, Bossers A, van Zijderveld FG, van Keulen LJ. Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep. BMC Vet Res. 2006;2:19. doi:10.1186/1746-6148-2-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Langevin C, Andreoletti O, Le Dur A, Laude H, Beringue V. Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model. Neurobiol Dis. 2011;41:219–25. doi:10.1016/j.nbd.2010.09.010.View ArticlePubMedGoogle Scholar
- Lasmezas CI, Fournier JG, Nouvel V, Boe H, Marce D, Lamoury F, et al. Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt-- Jakob disease: implications for human health. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:4142–7. doi:10.1073/pnas.041490898.PubMed CentralView ArticlePubMedGoogle Scholar
- Le Dur A, Beringue V, Andreoletti O, Reine F, Lai TL, Baron T, et al. A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:16031–6. doi:10.1073/pnas.0502296102.PubMed CentralView ArticlePubMedGoogle Scholar
- Li J, Browning S, Mahal SP, Oelschlegel AM, Weissmann C. Darwinian evolution of prions in cell culture. Science. 2010;327:869–72. doi:10.1126/science.1183218.PubMed CentralView ArticlePubMedGoogle Scholar
- Li J, Mahal SP, Demczyk CA, Weissmann C. Mutability of prions. EMBO Rep. 2011;12:1243–50. doi:10.1038/embor.2011.191.PubMed CentralView ArticlePubMedGoogle Scholar
- Mahal SP, Browning S, Li J, Suponitsky-Kroyter I, Weissmann C. Transfer of a prion strain to different hosts leads to emergence of strain variants. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:22653–8. doi:10.1073/pnas.1013014108.PubMed CentralView ArticlePubMedGoogle Scholar
- Moda F, Suardi S, Di Fede G, Indaco A, Limido L, Vimercati C, et al. MM2-thalamic Creutzfeldt-Jakob disease: neuropathological, biochemical and transmission studies identify a distinctive prion strain. Brain Pathol. 2012;22:662–9. doi:10.1111/j.1750-3639.2012.00572.x.View ArticlePubMedGoogle Scholar
- Moudjou M, Sibille P, Fichet G, Reine F, Chapuis J, Herzog L, et al. Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification. MBio. 2014;5:e00829–00813. doi:10.1128/mBio.00829-13.PubMed CentralView ArticleGoogle Scholar
- Neale MH, Mountjoy SJ, Edwards JC, Vilette D, Laude H, Windl O, et al. Infection of cell lines with experimental and natural ovine scrapie agents. Journal of virology. 2010;84:2444–52. doi:10.1128/JVI.01855-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Nicot S, Bencsik A, Morignat E, Mestre-Frances N, Perret-Liaudet A, Baron T. Differentiation of prions from L-type BSE versus sporadic Creutzfeldt-Jakob disease. Emerging infectious diseases. 2012;18:2028–31. doi:10.3201/eid1812.120342.PubMed CentralView ArticlePubMedGoogle Scholar
- Ojosnegros S, Perales C, Mas A, Domingo E. Quasispecies as a matter of fact: viruses and beyond. Virus Res. 2011;162:203–15. doi:10.1016/j.virusres.2011.09.018.View ArticlePubMedGoogle Scholar
- Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, et al. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Annals of neurology. 1999;46:224–33.View ArticlePubMedGoogle Scholar
- Pattison IH, Jones KM. Modification of a strain of mouse-adapted scrapie by passage through rats. Res Vet Sci. 1968;9:408–10.PubMedGoogle Scholar
- Pattison IH, Millson GC. Scrapie produced experimentally in goats with special reference to the clinical syndrome. J Comp Pathol. 1961;71:101–9.View ArticlePubMedGoogle Scholar
- Polymenidou M, Stoeck K, Glatzel M, Vey M, Bellon A, Aguzzi A. Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease. Lancet Neurol. 2005;4:805–14. doi:10.1016/S1474-4422(05)70225-8.View ArticlePubMedGoogle Scholar
- Prusiner SB. Prions. Proceedings of the National Academy of Sciences of the United States of America. 1998;95:13363–83.PubMed CentralView ArticlePubMedGoogle Scholar
- Prusiner SB, Cochran SP, Groth DF, Downey DE, Bowman KA, Martinez HM. Measurement of the scrapie agent using an incubation time interval assay. Annals of neurology. 1982;11:353–8. doi:10.1002/ana.410110406.View ArticlePubMedGoogle Scholar
- Puoti G, Giaccone G, Rossi G, Canciani B, Bugiani O, Tagliavini F. Sporadic Creutzfeldt-Jakob disease: co-occurrence of different types of PrP(Sc) in the same brain. Neurology. 1999;53:2173–6.View ArticlePubMedGoogle Scholar
- Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001;411:810–3. doi:10.1038/35081095.View ArticlePubMedGoogle Scholar
- Schoch G, Seeger H, Bogousslavsky J, Tolnay M, Janzer RC, Aguzzi A, et al. Analysis of prion strains by PrPSc profiling in sporadic Creutzfeldt-Jakob disease. PLoS Med. 2006;3, e14. doi:10.1371/journal.pmed.0030014.PubMed CentralView ArticlePubMedGoogle Scholar
- Scott MR, Groth D, Tatzelt J, Torchia M, Tremblay P, DeArmond SJ, et al. Propagation of prion strains through specific conformers of the prion protein. Journal of virology. 1997;71:9032–44.PubMed CentralPubMedGoogle Scholar
- Shikiya RA, Ayers JI, Schutt CR, Kincaid AE, Bartz JC. Coinfecting prion strains compete for a limiting cellular resource. Journal of virology. 2010;84:5706–14. doi:10.1128/JVI.00243-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Tixador P, Herzog L, Reine F, Jaumain E, Chapuis J, Le Dur A, et al. The physical relationship between infectivity and prion protein aggregates is strain-dependent. PLoS Pathog. 2010;6, e1000859. doi:10.1371/journal.ppat.1000859.PubMed CentralView ArticlePubMedGoogle Scholar
- Uro-Coste E, Cassard H, Simon S, Lugan S, Bilheude JM, Perret-Liaudet A, et al. Beyond PrP9res) type 1/type 2 dichotomy in Creutzfeldt-Jakob disease. PLoS pathogens. 2008;4, e1000029. doi:10.1371/journal.ppat.1000029.PubMed CentralView ArticleGoogle Scholar
- Vilette D, Andreoletti O, Archer F, Madelaine MF, Vilotte JL, Lehmann S, et al. Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein. Proc Natl Acad Sci U S A. 2001;98:4055–9. doi:10.1073/pnas.061337998.PubMed CentralView ArticlePubMedGoogle Scholar
- Vilotte JL, Soulier S, Essalmani R, Stinnakre MG, Vaiman D, Lepourry L, et al. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp. Journal of virology. 2001;75:5977–84. doi:10.1128/JVI.75.13.5977-5984.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Weissmann C. Mutation and selection of prions. PLoS pathogens. 2012;8, e1002582. doi:10.1371/journal.ppat.1002582.PubMed CentralView ArticlePubMedGoogle Scholar
- Weissmann C, Li J, Mahal SP, Browning S. Prions on the move. EMBO Rep. 2011;12:1109–17. doi:10.1038/embor.2011.192.PubMed CentralView ArticlePubMedGoogle Scholar
- Will RG. Acquired prion disease: iatrogenic CJD, variant CJD, kuru. Br Med Bull. 2003;66:255–65.View ArticlePubMedGoogle Scholar