Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Change in the molecular properties of CH1641 prions after transmission to wild-type mice: Evidence for a single strain.

AIM: CH1641 was discovered in 1970 as a scrapie isolate that was unlike all other classical strains of scrapie isolated so far. We performed bio-assays of CH1641 in mice in order to further characterise this specific isolate. METHODS: We inoculated the original CH1641 isolate into ovine and bovine prion protein (PrP) transgenic mice as well as wild-type mice. In addition, we performed cross- and back passages between the various mouse lines to examine if one identical prion strain was isolated in all mouse lines or whether multiple prion strains exist in CH1641. RESULTS: We report the first successful transmission of CH1641 to wild-type RIII mice and via RIII mice to wild-type VM mice. Unexpectedly, analysis of the protease-resistant prion protein (PrPres ) in wild-type mice showed a classical scrapie banding pattern differing from the banding pattern of the original CH1641 isolate. Cross- and back passages of CH1641 between the various mouse lines confirmed that the same prion strain had been isolated in all mouse lines. CONCLUSIONS: The CH1641 isolate consists of a single prion strain but its molecular banding pattern of PrPres differs between wild-type mice and PrP transgenic mice. Consequently, molecular banding patterns of PrPres should be used with caution in strain typing since they do not solely depend on the properties of the prion strain but also on the host prion protein.

Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie.

OBJECTIVE: Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. METHODS: Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. RESULTS: Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrPSc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. CONCLUSION: Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals' scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.

Increased Gal-3 Mediates Microglia Activation and Neuroinflammation via the TREM2 Signaling Pathway in Prion Infection.

Galectin 3 (Gal-3) is one of the major elements for activating microglia and mediating neuroinflammation in some types of neurodegenerative diseases. However, its role in the pathogenesis of prion disease is seldom addressed. In this study, markedly increased brain Gal-3 was identified in three scrapie-infected rodent models at the terminal stage. The increased Gal-3 was mainly colocalized with the activated microglia. Coincidental with the increased brain Gal-3 in prion-infected animals, the expression of brain trigger receptor expressed in myeloid cell 2 (TREM2), one of the Gal-3 receptors, and some components in the downstream pathway also significantly increased, whereas Toll-like receptor 4 (TLR4), another Gal-3 receptor, and the main components in its downstream signaling were less changed. The increased Gal-3 signals were distributed at the areas with PrPSc deposit but looked not to colocalize directly with PrPSc/PrP signals. Similar changing profiles of Gal-3, the receptors TREM2 and TLR4, as well as the proteins in the downstream pathways were also observed in prion-infected cell line SMB-S15. Removal of PrPSc replication in SMB-S15 cells reversed the upregulation of cellular Gal-3, TREM2, and the relevant proteins. Moreover, we presented data for interactions of Gal-3 with TREM2 and with TLR4 morphologically and molecularly in the cultured cells. Stimulation of prion-infected cells or their normal partner cells with recombinant mouse Gal-3 in vitro induced obvious responses for activation of TREM2 signaling and TLR4 signaling. Our data here strongly indicate that prion infection or PrPSc deposit induces remarkably upregulated brain Gal-3, which is actively involved in the microglia activation and neuroinflammation mainly via TREM2 signaling.

Radotinib Decreases Prion Propagation and Prolongs Survival Times in Models of Prion Disease.

The conversion of cellular prion protein (PrPC) into pathogenic prion isoforms (PrPSc) and the mutation of PRNP are definite causes of prion diseases. Unfortunately, without exception, prion diseases are untreatable and fatal neurodegenerative disorders; therefore, one area of research focuses on identifying medicines that can delay the progression of these diseases. According to the concept of drug repositioning, we investigated the efficacy of the c-Abl tyrosine kinase inhibitor radotinib, which is a drug that is approved for the treatment of chronic myeloid leukemia, in the treatment of disease progression in prion models, including prion-infected cell models, Tga20 and hamster cerebellar slice culture models, and 263K scrapie-infected hamster models. Radotinib inhibited PrPSc deposition in neuronal ZW13-2 cells that were infected with the 22L or 139A scrapie strains and in cerebellar slice cultures that were infected with the 22L or 263K scrapie strains. Interestingly, hamsters that were intraperitoneally injected with the 263K scrapie strain and intragastrically treated with radotinib (100 mg/kg) exhibited prolonged survival times (159 ± 28.6 days) compared to nontreated hamsters (135 ± 9.9 days) as well as reduced PrPSc deposition and ameliorated pathology. However, intraperitoneal injection of radotinib exerted a smaller effect on the survival rate of the hamsters. Additionally, we found that different concentrations of radotinib (60, 100, and 200 mg/kg) had similar effects on survival time, but this effect was not observed after treatment with a low dose (30 mg/kg) of radotinib. Interestingly, when radotinib was administered 4 or 8 weeks after prion inoculation, the treated hamsters survived longer than the vehicle-treated hamsters. Additionally, a pharmacokinetic assay revealed that radotinib effectively crossed the blood-brain barrier. Based on our findings, we suggest that radotinib is a new candidate anti-prion drug that could possibly be used to treat prion diseases and promote the remission of symptoms.

Full-length prion protein incorporated into prion aggregates is a marker for prion strain-specific destabilization of aggregate structure following cellular uptake.

Accumulation of insoluble aggregates of infectious, partially protease-resistant prion protein (PrPD) generated via the misfolding of protease sensitive prion protein (PrPC) into the same infectious conformer, is a hallmark of prion diseases. Aggregated PrPD is taken up and degraded by cells, a process likely involving changes in aggregate structure that can be monitored by accessibility of the N-terminus of full-length PrPD to cellular proteases. We therefore tracked the protease sensitivity of full-length PrPD before and after cellular uptake for two murine prion strains, 22L and 87V. For both strains, PrPD aggregates were less stable following cellular uptake with increased accessibility of the N-terminus to cellular proteases across most aggregate sizes. However, a limited size range of aggregates was able to better protect the N-termini of full-length PrPD, with the N-terminus of 22L-derived PrPD more protected than that of 87V. Interestingly, changes in aggregate structure were associated with minimal changes to the protease-resistant core of PrPD. Our data show that cells destabilize the aggregate quaternary structure protecting PrPD from proteases in a strain-dependent manner, with structural changes exposing protease sensitive PrPD having little effect on the protease-resistant core, and thus conformation, of aggregated PrPD.

Synthesis, structural characterization and study of antioxidant and anti-PrPSc properties of flavonoids and their rhenium(I)-tricarbonyl complexes.

This study aims at the synthesis and initial biological evaluation of novel rhenium-tricarbonyl complexes of 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,7,4΄-trihydroxyflavone (resokaempferol), 5,7-dihydroxyflavone (chrysin) and 4΄,5,7-trihydroxyflavonone (naringenin) as neuroprotective and anti-PrP agents. Resokaempferol was synthesized from 2,2΄,4-trihydroxychalcone by H2O2/NaOH. The rhenium-tricarbonyl complexes of the type fac-[Re(CO)3(Fl)(sol)] were synthesized by reacting the precursor fac-[Re(CO)3(sol)3]+ with an equimolar amount of the flavonoids (Fl) quercetin, resokaempferol, chrysin and naringenin and the solvent (sol) was methanol or water. The respective Re-flavonoid complexes were purified by semi-preparative HPLC and characterized by spectroscopic methods. Furthermore, the structure of Re-chrysin was elucidated by X-ray crystallography. Initial screening of the neuroprotective properties of these compounds included the in vitro assessment of the antioxidant properties by the DPPH assay as well as the anti-lipid peroxidation of linoleic acid in the presence of AAPH and their ability to inhibit soybean lipoxygenase. From the above studies, it was concluded that the complexes' properties are mainly correlated with the structural characteristics and the presence of the flavonoids. The flavonoids and their respective Re-complexes were also tested in vitro for their ability to inhibit the formation and aggregation of the amyloid-like abnormal prion protein, PrPSc, by employing the real-time quaking-induced conversion assay with recombinant PrP seeded with cerebrospinal fluid from patients with Creutzfeldt-Jakob disease. All the compounds blocked de novo abnormal PrP formation and aggregation.

Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism.

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

Role of sialylation of N-linked glycans in prion pathogenesis.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30 years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication.

Cholesterol and its reciprocal association with prion infection.

Prion diseases are incurable, infectious and fatal neurodegenerative diseases that affect both humans and animals. The pathogenesis of prion disease involves the misfolding of the cellular prion protein, PrPC, to a disease-causing conformation, PrPSc, in the brain. The exact mechanism of conversion of PrPC to PrPSc is not clear; however, there are numerous studies supporting that this process of misfolding requires the association of PrPC with lipid raft domains of the plasma membrane. An increase in the cellular cholesterol content with prion infection has been observed in both in vivo and in vitro studies. As cholesterol is critical for the formation of lipid rafts, on the one hand, this increase may be related to, or aiding in, the process of prion conversion. On the other hand, increased cholesterol levels may affect neuronal viability. Here, we discuss current literature on the underlying mechanisms and potential consequences of elevated neuronal cholesterol in prion infection and advancements in prion disease therapeutics targeting brain cholesterol homeostasis.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep.

The agent of scrapie is resistant to most chemical and physical methods of inactivation. Prions bind to soils, metals, and various materials and persist in the environment confounding the control of prion diseases. Most methods of prion inactivation require severe conditions such as prolong exposure to sodium hypochlorite or autoclaving, which may not be suitable for field conditions. We evaluated the efficacy of a combinatorial approach to inactivation of US scrapie strain x124 under the mild conditions of treating scrapie-affected brain homogenate with sodium percarbonate (SPC), sodium dodecyl sulfate (SDS), or in combination followed by proteinase K (PK) digestion at room temperature. Western blot analysis of treated brain homogenate demonstrates partial reduction in PrPSc immunoreactivity. Genetically susceptible VRQ/ARQ Suffolk sheep were oronasally inoculated with 1 g of SPC (n = 1), SDS (n = 2), SDS + PK (n = 2), and SPC + SDS + PK (n = 4) treated brain homogenate. Sheep were assessed daily for clinical signs, euthanized at the development of clinical disease, and tissues were assessed for accumulation of PrPSc. Scrapie status in all sheep was determined by western blot, enzyme immunoassay, and immunohistochemistry. Mean incubation periods (IPs) for SPC (11.9 months, 0% survival), SDS (12.6 months, 0% survival), SDS + PK (14.0 months, 0% survival), and SPC + SDS + PK (12.5 months, 25% survival) were increased compared to positive control sheep (n = 2, 10.7 months, 0% survival) by 1.2, 1.9, 3.3, and 1.8 months, respectively. Treatment did not influence PrPSc accumulation and distribution at the clinical stage of disease. Differences in mean IPs and survival indicates partial but not complete reduction in scrapie infectivity.

Chronological Changes in the Expression Pattern of Hippocampal Prion Proteins During Disease Progression in Sporadic Creutzfeldt-Jakob Disease MM1 Subtype.

The differential effects of sporadic Creutzfeldt-Jakob disease (sCJD) on the hippocampus and other neocortical areas are poorly understood. We aimed to reveal the histological patterns of cellular prion protein (PrPC) and abnormal prion protein (PrPSc) in hippocampi of sCJD patients and normal controls (NCs). Our study examined 18 postmortem sCJD patients (MM1, 14 cases; MM1 + 2c, 3 cases; MM1 + 2t, 1 case) and 12 NCs. Immunohistochemistry was conducted using 4 primary antibodies, of which 3 targeted the N-terminus of the prion protein (PrP), and 1 (EP1802Y) targeted the C-terminal domain. PrPC expression was abundant in the hippocampus of NCs, and the distribution of PrPC at CA3/4 was reminiscent of synaptic complexes. In sCJD cases with a disease history of <2 years, antibodies against the N-terminus could not detect synapse-like PrP expression at CA4; however, EP1802Y could characterize the synapse-like expression. PrPSc accumulation and spongiform changes became evident after 2 years of illness, when PrPSc deposits were more noticeably detected by N-terminal-specific antibodies. Our findings highlighted the chronology of histopathological alterations in the CA4 region in sCJD patients.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages.

Objective: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages. Methods: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs). Results: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages. Conclusion: Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263 K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Early Stages of RNA-Mediated Conversion of Human Prions.

Prion diseases are characterized by the conversion of prion proteins from a PrPC fold into a disease-causing PrPSC form that is self-replicating. A possible agent to trigger this conversion is polyadenosine RNA, but both mechanism and pathways of the conversion are poorly understood. Using coarse-grained molecular dynamic simulations we study the time evolution of PrPC over 600 µs. We find that both the D178N mutation and interacting with polyadenosine RNA reduce the helicity of the protein and encourage formation of segments with strand-like motifs. We conjecture that these transient ß-strands nucleate the conversion of the protein to the scrapie conformation PrPSC.

Subclinical infection occurs frequently following low dose exposure to prions by blood transfusion.

Infectious prion diseases have very long incubation periods, and the role that subclinical infections play in transmission, persistence and re-emergence of these diseases is unclear. In this study, we used a well-established model of vCJD (sheep experimentally infected with bovine spongiform encephalopathy, BSE) to determine the prevalence of subclinical infection following exposure by blood transfusion from infected donors. Many recipient sheep survived for years post-transfusion with no clinical signs and no disease-associated PrP (PrPSc) found in post mortem tissue samples by conventional tests. Using a sensitive protein misfolding cyclic amplification assay (PMCA), we found that the majority of these sheep had detectable PrPSc in lymph node samples, at levels approximately 105-106 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrPSc in other tissues, including brain, but not in blood samples. The results demonstrate that subclinical infection is a frequent outcome of low dose prion infection by a clinically relevant route for humans (blood transfusion). The long term persistence of low levels of infection has important implications for prion disease control and the risks of re-emergent infections in both humans and animals.