Aileen Boyle

Strain characterization of natural sheep scrapie and comparison with BSE.

Scrapie was transmitted to mice from ten sheep, collected in the UK between 1985 and 1994. As in previous natural scrapie transmissions, the results varied between scrapie sources in terms of the incidence of disease, incubation periods and neuropathology in challenged mice. This contrasted with the uniformity seen in transmissions of BSE to mice. The scrapie and BSE isolates were characterized further by serial passage in mice. Different TSE strains were isolated from each source according to the Sinc or PrP genotype of the mouse used for passage. The same two mouse-passaged strains, 301C and 301V, were isolated from each of three BSE sources. Despite the variation seen in the primary transmissions of scrapie, relatively few mouse-passaged scrapie strains were isolated and these were distinct from the BSE-derived strains. The ME7 scrapie strain, which has often been isolated from independent sheep sources in the past, was identified in isolates from four of the sheep. However, a new distinct strain, 221C, was derived from a further four scrapie sheep. These results suggest that there is agent strain variation in natural scrapie in sheep and that the spectrum of strains present may have changed over the last 20 years. The tested sample is too small to come to any conclusions about whether the BSE strain is present in sheep, but the study provides a framework for further more extensive studies.

Variant CJD: 18 years of research and surveillance

It is now 18 years since the first identification of a case of vCJD in the UK. Since that time, there has been much speculation over how vCJD might impact human health. To date there have been 177 case reports in the UK and a further 51 cases worldwide in 11 different countries. Since establishing that BSE and vCJD are of the same strain of agent, we have also shown that there is broad similarity between UK and non-UK vCJD cases on first passage to mice. Transgenic mouse studies have indicated that all codon 129 genotypes are susceptible to vCJD and that genotype may influence whether disease appears in a clinical or asymptomatic form, supported by the appearance of the first case of potential asymptomatic vCJD infection in a PRNP 129MV patient. Following evidence of blood transfusion as a route of transmission, we have ascertained that all blood components and leucoreduced blood in a sheep model of vCJD have the ability to transmit disease. Importantly, we recently established that a PRNP 129MV patient blood recipient with an asymptomatic infection and limited PrPSc deposition in the spleen could readily transmit disease into mice, demonstrating the potential for peripheral infection in the absence of clinical disease. This, along with the recent appendix survey which identified 16 positive appendices in a study of 32 441 cases, underlines the importance of continued CJD surveillance and maintaining control measures already in place to protect human health.

The Glycosylation Status of PrPC Is a Key Factor in Determining Transmissible Spongiform Encephalopathy Transmission between Species

ABSTRACT The risk of transmission of transmissible spongiform encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. A transmission barrier between species often prevents infection of a new host with a TSE agent. Nonetheless, some TSE agents are able to cross this barrier and infect new species, with devastating consequences. The host PrPC misfolds during disease pathogenesis and has a major role in controlling the transmission of agents between species, but sequence compatibility between host and agent PrPC does not fully explain host susceptibility. PrPC is posttranslationally modified by the addition of glycan moieties which have an important role in the infectious process. Here, we show in vivo that glycosylation of the host PrPC has a significant impact on the transmission of TSE between different host species. We infected mice carrying different glycosylated forms of PrPC with two human agents (sCJDMM2 and vCJD) and one hamster strain (263K). The absence of glycosylation at both or the first PrPC glycosylation site in the host results in almost complete resistance to disease. The absence of the second site of N-glycan has a dramatic effect on the barrier to transmission between host species, facilitating the transmission of sCJDMM2 to a host normally resistant to this agent. These results highlight glycosylation of PrPC as a key factor in determining the transmission efficiency of TSEs between different species. IMPORTANCE The risks of transmission of TSE between different species are difficult to predict due to a lack of knowledge over the mechanisms of disease transmission; some strains of TSE are able to cross a species barrier, while others do not. The host protein, PrPC, plays a major role in disease transmission. PrPC undergoes posttranslational glycosylation, and the addition of these glycans may play a role in disease transmission. We infected mice that express different forms of glycosylated PrPC with three different TSE agents. We demonstrate that changing the glycosylation status of the host can have profound effects on disease transmission, changing host susceptibility and incubation times. Our results show that PrPC glycosylation is a key factor in determining risks of TSE transmission between species.

Variably Protease-Sensitive Prionopathy, a Unique Prion Variant with Inefficient Transmission Properties

Transmission properties of this prion disease are biologically distinct, and the disease has limited potential for human-to-human transmission.

TSE Strain Typing in Mice

It has been known for many years that transmissible spongiform encephalopathy (TSE) agents exhibit strain variation [1], a phenomenon that has been studied most extensively in experimental mouse models. Numerous distinct TSE strains have been isolated in mice from a range of scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) sources. The methods used for TSE strain discrimination are based on simple observations of disease characteristics, the most useful being the length of the incubation period of the disease and the type and extent of the pathological changes seen hi the brains of infected animals [24]. Formal strain typing protocols in mice, based on incubation periods and neuropathology, have been used extensively as tools in basic research, for example in studies exploring the nature of TSE agents.

The cellular form of the prion protein PrPC is processed to varying degrees in different species and PRNP genotypes

Creutzfeldt-Jakob disease (CJD) is characterized by a variety of symptoms including rapidly progressive neurocognitive decline, ataxic gait, and myoclonus. Sporadic CJD (sCJD) has a median disease duration of 6 months. Based on the prion protein gene (PRNP) codon 129 polymorphism and prion protein type, seven different molecular subtypes of sCJD have been identified (MM1, MM2-C, MM2-T, MV1, MV2, VV1, VV2). The VV1 subtype is the rarest molecular subtype of sCJD (only about 1% of cases). To date, reported cases of sCJD VV1 have been characterized by young age at disease onset, an increased length of disease duration, and progressive neurocognitive degeneration. We describe a unique case of sCJD VV1 with older age at disease onset and short illness duration. VV1 is a challenging subtype to diagnose and this report adds further complexity and variation to its clinical characterization. Although PRNP codon 129 polymorphism and prion protein type exert a large influence on clinical and neuropathologic phenomenology in sCJD, other factors remain unknown and require further study. P.02: A transfectant RK13 cell line permissive to caprine scrapie prion infection

Strain Typing of Prion Diseases Using In Vivo Mouse Models

Transmissible spongiform encephalopathies (TSE) or prion diseases exhibit strain variation, a phenomenon that has been studied extensively in mouse bioassays. Despite the introduction of many rapid in vitro systems, bioassays remain a key tool in defining prion strains and their ability to transmit disease in vivo. Prion strains can be characterized by a range of phenotypic characteristics such as incubation period, vacuolar pathology, and distribution of the abnormal form of PrP following experimental transmission of the agent into a panel of mice (transgenic or wild type). Interpretation of these characteristics requires considerable experience and an understanding of the procedures used to define them. This chapter reviews the techniques used in strain typing of prion diseases from inoculum preparation and pathological studies to data interpretation alongside an extensive troubleshooting guide.

Similarities of Variant Creutzfeldt-Jakob Disease Strain in Mother and Son in Spain to UK Reference Case

We investigated transmission characteristics of variant Creutzfeldt-Jakob disease in a mother and son from Spain. Despite differences in patient age and disease manifestations, we found the same strain properties in these patients as in UK vCJD cases. A single strain of agent appears to be responsible for all vCJD cases to date.

Variant CJD: Lessons in public health

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant PrP

Assessing the disease-modifying role of TREM2 in a prion model of neurodegeneration

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant PrP