Viral Properties of Scrapie

ALTHOUGH scrapie agent has many unusual features^1–4, some aspects of its behaviour are analogous to viral properties⁵. The manner in which the agent invades the nervous system after peripheral inoculation immediately recalls such typically neurotropic viruses as poliomyelitis or rabies. After inoculation into the fore-limb changes occur first in the cervical cord; following injection into the leg they appear first in the lumbar cord⁶. We now report another way in which scrapie behaves as a classical virus.     

Scrapie resistance in ARQ sheep

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.     

Environmental Sources of Scrapie Prions

Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.Prion diseases are fatal neurological disorders. The archetypal prion disease is scrapie in sheep, and in the last few decades novel prion diseases have emerged in a range of species, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and variant Creutzfeldt-Jakob disease (vCJD) in humans. The “protein-only” hypothesis dictates that a pathological isoform, PrP^Sc, of the cellular prion protein (PrP^C) constitutes the infectious agent, or prion (13). A wide range of tissues from CWD- and scrapie-affected animals contain PrP^Sc, and affected animals have been shown to excrete or secrete prions in milk, saliva, urine, and feces (2, 3, 6, 7, 8, 10, 16). This finding led to the hypothesis that infectivity resides in the environment, thus explaining the facile transmission of CWD and scrapie. In support of this hypothesis, CWD infectivity has been transmitted from a combination of exposed bedding, water, and food of captive animals (9), and CWD PrP^Sc has been detected within a single environmental water sample (11).Environmental prions are likely to be present at very low levels. The most sensitive method available for the detection of PrP^Sc is serial protein-misfolding cyclic amplification (sPMCA) (1). This technique was pioneered by Soto and colleagues (15), has been used for the amplification of scrapie (17), CWD (4), and vCJD (5) within their natural hosts, and has facilitated the detection of prions within ovine milk (6) and saliva (7) and within cervine urine (4). Here we investigated sources of environmental scrapie prions by applying sPMCA to samples taken from a range of surfaces that were accessible to animals on a farm where scrapie is endemic.Environmental samples were taken from the Veterinary Laboratories Agency, United Kingdom, farm where natural scrapie is endemic, with a high incidence since 1996. Sheep within the flock were exposed to the scrapie agent by natural routes of transmission. Control samples were taken from a farm (ADAS, United Kingdom) that houses a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions. Environmental swabs were taken by wetting foam swabs (Edson Electronics, Northumberland, United Kingdom) in sterile water and then gently swabbing five times in both directions across a surface approximately 10 cm by 2 cm. Two swabs were taken from each area, and all samples were stored at −80^°C. A total of nine environmental samples from a scrapie-affected farm and a scrapie-free farm were analyzed by sPMCA.Two swabs taken from each area were thawed to room temperature and placed in a single container to which 6 ml of 150 mM PO₄ buffer plus 0.5% (vol/vol) Nonidet P-40 and 0.5% (wt/vol) sodium deoxycholate were added. The container was rotated for 2 h. Prions released into this buffer were precipitated on silicon dioxide and then eluted in 200 μl of 0.1% (wt/vol) sodium dodecyl sulfate. Ten microliters of the eluate was amplified within PCR tubes by sPMCA exactly as previously described (7). Samples from both a scrapie-exposed environment and a non-scrapie-exposed environment were analyzed concurrently within the same run on the same sonicator. Each extract was amplified at least in triplicate within a single run and then analyzed by Western blotting (14).Samples from four metal surfaces from an indoor pen occupied by sheep for a few days each week—a gate, a water trough, a feed trough, and penning—were analyzed. Samples from an outdoor environment that had contained sheep 20 days previously—a metal fence, a metal gate, a metal water trough, a plastic post where sheep frequently scratched, and a wooden fence post (Table ​(Table1)—were1)—were also analyzed. After 8 rounds of amplification, PrP^Sc was detected in all samples with the exceptions of the outdoor water trough and gate (Figure ​(Figure11 and Table ​Table1).1). Equivalent samples from a farm housing a scrapie-free flock were also analyzed, and PrP^Sc was not amplified even after 10 rounds of sPMCA. For indoor surfaces from the scrapie-affected farm, 83% of the sPMCA reactions were positive (n = 12), and 0% were positive for equivalent samples from a scrapie-free farm (n = 24). Similarly, 27% of analyses were positive for samples from outdoor surfaces (n = 15), and again no prion was amplified from equivalent samples taken from a scrapie-free farm (n = 30). For comparison of the percentages of positive sPMCA reactions for different cohorts of samples, data were set up as 2 by 2 contingency tables, and Fisher''s exact test (one-tailed) was applied to derive P values. Overall, prions were significantly more likely to be present in the scrapie-affected farm on indoor (P < 0.001) and outdoor (P = 0.009) surfaces. Analyses of all samples were carried out in two independent experiments that gave equivalent results.Open in a separate windowFIG. 1.Amplification of prions from environmental samples. Prions were extracted from swabs taken of surfaces from a scrapie-free farm or a farm where scrapie is endemic. Swabs were taken from a wooden post (1), a plastic scratching post (2), and the following metal surfaces: fencing (3), gate (4 and 6), pen (5), feed trough (7), and water trough (8 and 9). Samples 1, 2, 3, 4, and 8 were taken from outdoor surfaces and 5, 6, 7, and 9 from indoor surfaces. Extracts were used as seeds for 8 rounds of sPMCA. Products were digested with proteinase K and analyzed by Western blotting. PrP was detected with monoclonal antibodies SHA31 and P4. Molecular-weight markers are shown.

TABLE 1.

sPMCA of prions found in the environment^a

Isolation of Scrapie Agent from the Placenta of Sheep with Natural Scrapie in Japan

A five-month-pregnant Suffolk sheep histologically diagnosed as spontaneous scrapie was studied. Western blot analysis was performed with rabbit serum against the sheep scrapie-associated fibrils (SAF). In the proteinase K (pk)-treated parental brain and spleen samples, three major bands (15 K, 18 K, and 23 K) were detected. These major bands were not detected from the placenta. Infectious agents were isolated in mice from the brain samples but not from the placental homogenates. In another case of a three-month-pregnant Corriedale sheep without any clinical sign of, but histologically diagnosed as scrapie, was also studied in a similar approach. In the parental brain samples, three major bands (15 K, 18 K and 23 K) were detected. SAF protein was not detected in the parental spleen and placenta. No bands reactive with the antiserum were detected in any other samples from the fetal brain and spleen in both cases. However, infectious agents were isolated in mice from both brain and placental homogenates. Since the placenta is an important site of natural infection, it is worthwhile to study these tissues for the epidemiological study of scrapie infection.     

Scrapie in Sheep in Sweden

In 1986 scrapie was diagnosed in 2 ewes of Swedish landrace (finn sheep) from a herd south of Stockholm (Carlsson et al 1986). As the diagnosis was based on clinical histo-ry and patho-anathomical changes only, inoculation tests in mice and goats were per-formed to try to verify the diagnosis.     

Scrapie I. Transmission and Pathogenesis

Homogenized brain, extracts, and residues of brain from normal and scrapie sheep were inoculated into 116 sheep. Of 72 sheep inoculated with scrapie material 27 developed the disease, whereas four of 44 inoculated with ;normal' brain material showed symptoms similar to those of scrapie. The scrapie agent survived extraction for 18 hours with diethyl ether followed by water for 24 hours. Similarly the agent survived extraction in a Soxhlet apparatus with 95 per cent ethanol containing 2.0 per cent 4 M HC1 for 24 hours. There was no evidence of demyelination in sections of brain and spinal cord supporting the view that allergic encephalomyelitis is not responsible for the scrapie symptoms. Liver function, as measured by the bromsulfalein test, remained normal until just before death. These results preclude that liver dysfunction contributes to the disease but do not exclude the possibilities of other metabolic derangements.     

Endocrinopathy in Mice affected with Scrapie

SCRAPIE is a protracted neurologic disease of sheep that is caused by a virus-like agent¹. The prominent neuropathologic lesions of scrapie are hypertrophy and proliferation of astrocytes, vacuolation of grey matter and progressive degeneration of neurones^2,3. The scrapie agent induces a similar disease in the mouse⁴. Characteristic clinical features include a waddling gait, reluctance to move, dullness, loss of condition and stiffness of the tail. Evidence of endocrinopathy was observed in two inbred strains of mice affected with scrapie. I shall describe pathologic changes in the reproductive organs of the Balb/c mouse and change in coat colour of the C57 black mouse.     

Scrapie prion protein contains a phosphatidylinositol glycolipid

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.     

Scrapie, ribosomal proteins and biological information

Consideration of the autocatalytic synthesis of ribosomal proteins leads to a criterion for the infectivity of a foreign proteinaceous species in terms of the biochemical rate constants governing the propagation of errors during the translation of genetic information in a model system. Evidence pertaining to the suggestion that scrapie and its analogues are caused by proteinaceous infectious agents (prions) which replicate by invading the translation process and altering ribosomal specificity is examined. It is found that anomalous aetiological features of scrapie infection are explained by the model. An analysis suggesting that the possibility of prion replication undermines the basis of current molecular biological theory is provided and it is concluded that the exclusive identification of biological information with nucleic acid sequences is unjustified.     

Is the Scrapie Agent a Viroid?

RECENT evidence indicates that the agent of the potato spindle tuber disease, previously thought to be a virus, is a replicating RNA with a molecular weight of about 50,000 (refs. 1 and 2). Because there seems to be no helper virus in the system¹, the RNA must rely for its replication principally on biosynthetic systems already functioning in the host. This, as well as the absence of virions, distinguishes the potato spindle tuber agent from all known viral agents. I suggested that such agents, which may include the agents of citrus exocortis³ and of chrysanthemum stunt⁴ diseases, should be called “viroids”¹. I now suggest that the agent of scrapie may also be a viroid.     

Absence of Eclipse Phase in Scrapie Mice

Field, Joyce and Keith¹ have claimed that the scrapie agent shows a viral characteristic by having an eclipse phase after being inoculated into the intracerebral region of mice. Three experiments, studying the accumulation of scrapie agent in spleen after intracerebral and intraperitoneal inoculation of mice, have not verified the conclusions of Field et al. A typical curve for the progression of scrapie activity after intracerebral inoculation (Fig. 1) shows no eclipse phase, nor was any observed in an experiment where the titre was examined every 2 days for the first 14 days after inoculation. Moreover, Field and his colleagues have not referred to similar studies with different results from theirs. Four independent groups of workers^2–6 have now examined the levels of scrapie activity in brain and spleen during the early stages after infection by three different routes and in none of these studies was there any clear indication of an eclipse phase in scrapie.     

Scrapie Pathogenesis in Subclinically Infected B-Cell-Deficient Mice

Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.     

Competition between Different Scrapie Agents in Mice

STRAINS of scrapie agent differ in a number of their biological properties, one of which is average incubation period^1,2. Incubation period is inversely proportional to dose for all known strains of scrapie and some differ so markedly that there is no overlap in their dose-incubation period curves. A large dose of one agent takes longer to produce the disease than the LD₅₀ dose of another agent³. Two such scrapie agents are 22A and 22C when compared by intracerebral injection of VM mice. Agent 22A has a much shorter incubation period than 22C in VM mice because they carry the p7 allele of the sine gene that controls scrapie incubation. It should therefore be possible to produce mixed infections in VM mice, but to ensure that the clinical disease is produced by the 22A agent. It can be checked that 22A causes the disease, rather than 22C, by examining the distribution of brain lesions². Interaction during pathogenesis between the two agents—competition or synergism—would be seen as a difference in the incubation period of 22A. We achieved mixed infection by injecting 22C first and 22A on a later occasion.     

Simple Method of Evaluating Scrapie in Mice

Mice in advanced scrapie characteristically clasp their hindlegs together. As the disease progresses into the clasping stage, mice exhibit a sequence of identifiable hindleg reactions which have categorized and assigned relative scores permitting objective, quantitative evaluation of disease.