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Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrP Sc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrP Sc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrP Sc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrP Sc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

The biological determinants of the phenotypic variation in sporadic Creutzfeldt-Jakob disease (sCJD) are unknown. To categorize sCJD cases, the prion protein (PrP) codon 129 genotype and the biochemical characteristics of the disease-associated form of PrP (PrP Sc ) can be combined to form six subgroups (MM1, MM2, MV1, MV2, VV1, and VV2). This classification largely correlates with the known variation in the clinical and pathological features of sCJD, with the MM1 and MV1 cases representing the \"classic\" phenotype of sCJD. To address how this classification relates to different strains of sCJD we have inoculated each subgroup of sCJD to a panel of mice expressing different forms of the human PRNP gene (129MM, 129VV, and 129MV). We have established that all subtypes are transmissible to at least one genotype of mouse, and both agent and host factors determine transmission efficiency and the form of PrP Sc deposited in the brain. Moreover, we have identified four distinct strains of sCJD using our in vivo strain typing panel.

Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for \"prion-like\" conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.

Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C) expression was specifically \"switched on\" or \"off\" only on FDC. We show that PrP(C)-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C)-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C) expression on FDC.

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion.

About the Authors: Abigail B. Diack Affiliation: The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, United Kingdom ORCID http://orcid.org/0000-0002-4293-3437 Robert G. Will Affiliation: National CJD Research and Surveillance Unit, University of Edinburgh, Edinburgh, United Kingdom Jean C. Manson * E-mail: Jean.manson@roslin.ed.ac.uk Affiliations The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, United Kingdom, The Centre for Dementia Prevention, University of Edinburgh, Edinburgh, United Kingdom, Edinburgh Neuroscience, University of Edinburgh, Edinburgh, United Kingdom ORCID http://orcid.org/0000-0002-0175-9890Citation: Diack AB, Will RG, Manson JC (2017) Public health risks from subclinical variant CJD. Introduction Variant Creutzfeldt-Jakob disease (vCJD) is a zoonotic prion disease thought to have been transmitted to humans through the consumption of food products contaminated with bovine spongiform encephalopathy (BSE) in the 1980s and/or early 1990s. (2006) Variant Creutzfeldt-Jakob disease: prion protein genotype analysis of positive appendix tissue samples from a retrospective prevalence study. (2009) Transmissions of variant Creutzfeldt-Jakob disease from brain and lymphoreticular tissue show uniform and conserved bovine spongiform encephalopathy-related phenotypic properties on primary and secondary passage in wild-type mice.

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.

Autolysosomal dysfunction and unstable microtubules are hallmarks of chronic neurodegenerative diseases associated with misfolded proteins. Investigation of impaired protein quality control and clearance systems could therefore provide an important avenue for intervention. To investigate this we have used a highly controlled model for protein aggregation, an in vitro prion system. Here we report that prion aggregates traffic via autolysosomes in the cytoplasm. Treatment with the natural polyamine spermine clears aggregates by enhancing autolysosomal flux. We demonstrated this by blocking the formation of mature autophagosomes resulting in accumulation of prion aggregates in the cytoplasm. Further we investigated the mechanism of spermine’s mode of action and we demonstrate that spermine increases the acetylation of microtubules, which is known to facilitate retrograde transport of autophagosomes from the cellular periphery to lysosomes located near the nucleus. We further report that spermine facilitates selective autophagic degradation of prion aggregates by binding to microtubule protein Tubb6. This is the first report in which spermine and the pathways regulated by it are applied as a novel approach towards clearance of misfolded prion protein and we suggest that this may have important implication for the broader family of protein misfolding diseases.

We investigated a clinical case of variant Creutzfeldt-Jakob Disease in a person heterozygous for methionine/valine at codon 129 of the prion protein gene and identified the same strain properties in variant Creutzfeldt-Jakob disease in methionine homozygous persons and in bovine spongiform encephalopathy. These results indicate no adaptation of the agent in a different genetic background.

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 (PrP Sc ) 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 PrP Sc in lymph node samples, at levels approximately 10 5 –10 6 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrP Sc 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.