The transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of neurodegenerative disorders which include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, and fatal familial insomnia in men, natural scrapie in sheep, goats and mufflons, transmissible mink encephalopathy in ranch-reared mink, chronic wasting disease of mule deer and elk, bovine spongiform encephalopathy or “mad cow disease” and its analogues in several exotic species of antelopes and wild felids in zoological gardens, and feline spongiform encephalopathy in domestic cats. This short review summarizes the history of the research to find the nature of the scrapie agent, especially as I have witnessed it unfolding before my eyes. I review the historical background of TSEs starting from the first description of scrapie in 1732. In 1957, the first prion disease in humans, kuru was described and its transmissibility was demonstrated in 1965 by seminal work of Gajdusek, Gibbs and colleagues, followed by transmission of CJD and then, GSS. In 1982, Stanley B. Prusiner formulated “prion hypothesis” which has dominated the field for the last 30 years. This theory had been recently extended to cover other neurodegenerations which are caused by misfolded proteins; these disease are called prionoids.

Following peripheral exposure, many transmissible spongiform encephalopathy (TSE) agents accumulate first in lymphoid tissues before spreading to the CNS (termed neuroinvasion) where they cause neurodegeneration. Early TSE agent accumulation upon follicular dendritic cells (FDCs) in lymphoid follicles appears critical for efficient neuroinvasion. Most clinical cases of variant Creutzfeldt-Jakob disease have occurred in young adults, although the reasons behind this apparent age-related susceptibility are uncertain. Host age has a significant influence on immune function. As FDC status and immune complex trapping is reduced in aged mice (600 days old), we hypothesized that this aging-related decline in FDC function might impair TSE pathogenesis. We show that coincident with the effects of host age on FDC status, the early TSE agent accumulation in the spleens of aged mice was significantly impaired. Furthermore, following peripheral exposure, none of the aged mice developed clinical TSE disease during their lifespans, although most mice displayed histopathological signs of TSE disease in their brains. Our data imply that the reduced status of FDCs in aged mice significantly impairs the early TSE agent accumulation in lymphoid tissues and subsequent neuroinvasion. Furthermore, the inefficient neuroinvasion in aged individuals may lead to significant levels of subclinical TSE disease in the population.

Prion strains are defined by their biological properties after transmission to wild-type mice, specifically by their incubation periods and patterns of vacuolar pathology ('lesion profiles'). Preliminary results from transmissions of variant Creutzfeldt-Jakob disease (vCJD) to wild-type mice provided the first compelling evidence for the close similarity of the vCJD agent to the agent causing bovine spongiform encephalopathy (BSE). Complete results from this investigation, including the transmission characteristics of vCJD from brain and peripheral tissues of 10 cases (after primary transmission and subsequent mouse-to-mouse passage), have now been analysed. All 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients were infected with the same strain of agent. Incubation periods suggested that infectious titres may be subject to regional variation within the brain. Comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Analysis of the protease-resistant prion protein (PrP(res)) by Western blotting from primary and subsequent passages in mice showed a glycosylation pattern closely resembling that of vCJD in humans, the so-called BSE 'glycoform signature'. Minor variations in PrP(res) fragment size were evident between mouse strains carrying different alleles of the gene encoding PrP both in primary transmissions and on further passages of vCJD brain. Overall, the results closely resembled those of previously reported transmissions of BSE in the same mouse strains, consistent with BSE being the origin of all of these vCJD cases.

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrPC, and the pathological isoform, PrPSc. In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (Kd) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrPC. Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrPSc following guanidinium denaturation.

Background The identification of transmission of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion has prompted investigation to establish whether there has been any alteration in the vCJD agent following this route of secondary transmission. Any increase in virulence or host adaptation would require a reassessment of the risk analyses relating to the possibility of a significant secondary outbreak of vCJD. Since there are likely to be carriers of the vCJD agent in the general population, there is a potential for further infection by routes such as blood transfusion or contaminated surgical instruments. Methodology We inoculated both wild-type and transgenic mice with material from the first case of transfusion associated vCJD infection. Principal Findings The strain transmission properties of blood transfusion associated vCJD infection show remarkable similarities to the strain of vCJD associated with transmission from bovine spongiform encephalopathy (BSE). Conclusions Although it has been hypothesized that adaptation of the BSE agent through secondary passage in humans may result in a greater risk of onward transmission due to an increased virulence of the agent for humans, our data presented here in two murine models suggest no significant alterations to transmission efficiency of the agent following human-to-human transmission of vCJD.

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

THE atypical form of scrapie in sheep, termed Nor98, was first described in clinically affected Norwegian sheep diagnosed from 1998 onwards (Benestad and others 2003). Nor98 is characterised by its atypical neuropathology and by the unusual banding pattern seen in Western blots of the pathological prion protein, PrPSc. Since Nor98 was first recognised, cases with similar features have been identified in several other countries, including the UK, mostly in the course of active surveillance of asymptomatic sheep (Buschmann and others 2004, De Bosschere and others 2004, Gavier-Widen and others 2004, Onnasch and others 2004, Orge and others 2004, Epstein and others 2005, Everest and others 2006). This short communication describes a clinical case of sheep scrapie with the distinguishing features of Nor98 that occurred in the UK in 1989. A Dorset horn sheep was presented to the Moredun Research Institute, Edinburgh, to confirm a clinical diagnosis of scrapie; records of the source flock and details of the clinical presentation are no longer available. Formalin-fixed and frozen brain samples were passed on to the Neuropathogenesis Unit for further investigation. The occurrence and incubation period of scrapie in sheep is controlled by the host gene encoding PrP (Hunter 1997). Many polymorphisms of this gene have been identified, but the major amino acid substitutions influencing scrapie are at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H). The PrP genotype of the Dorset horn sheep at these three codons was A136H154Q171/A136H154Q171 (AHQ/AHQ), a genotype that confers relative but not absolute resistance to ‘classical’ scrapie in the UK (Baylis and others 2004). However, cases of Nor98 scrapie in Norway have been significantly associated with the presence of the AHQ allele (Moum and others 2005). Neuropathological changes indicative of scrapie were seen predominantly in the cerebellum. This pathology consisted of a pronounced vacuolar degeneration of both the molecular and granular layers, accompanied by an abnormal accumulation of PrP (Fig 1); the changes were most severe in the granular layer. Milder pathological changes were seen in some brainstem nuclei, but areas adjacent to the obex, such as the dorsal motor nucleus of the vagus, were unaffected. The cerebral cortex showed only very mild pathology. This neuropathological distribution differs from that seen in most cases of classical scrapie, but, apart from the relative sparing of the cerebrum, resembles that seen in Nor98 scrapie (Benestad and others 2003). Protease-resistant PrPSc from the Dorset horn sheep was analysed by Western blotting and compared with PrPSc from cases of classical scrapie (in an ARQ/AHQ sheep) and Nor98 (in ARQ/AHQ and AHQ/AHQ sheep) (Fig 2). Brain tissues were homogenised in 100mM Tris-HCl (pH 7·4) with 2 per cent N-lauroylsarcosine sodium (Sarcosyl; Sigma), digested with proteinase K (200 μg/ml for one hour) and centrifuged at 20,000 g. The pellets were denatured in Laemmli buffer and loaded (3 mg tissue equivalents per lane) on 12 per cent Bis-Tris gel (Invitrogen). The blot was revealed by immunostaining with P4 monoclonal antibody. The Dorset horn isolate showed a PrPSc pattern very similar to the two Nor98 samples but very different from the classical scrapie sample. The Nor98-like pattern with P4 was characterised by the presence of multiple PrPSc fragments in the 12 to 30 kDa molecular weight range, with the 12 kDa fragments giving the strongest signal. In contrast, the classical scrapie isolate showed the typical three PrPSc bands, ranging from 18 to 30 kDa. The atypical phenotype seen in sheep with Nor98 scrapie suggests that they are infected with an unusual strain of scrapie. Scrapie strains can be characterised and compared by transmission studies in rodents, in which each strain produces a unique disease profile. The Nor98-like case described here was included in a study reported by Bruce and others (2002), in which transmissions to mice were attempted from 10 scrapieaffected sheep. The transmission from the Dorset horn sheep, referred to as SCR2 in the paper by Bruce and others (2002), was almost completely negative; this precluded any positive strain characterisation, as scrapie in general transmits poorly to mice. Brain tissue from the Dorset horn sheep was also inoculated into bank voles (Clethrionomys glareolus), together Veterinary Record (2007) 160, 665-666

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.