Transmissible Spongiform Encephalopathy Agents Research Articles

Transmissible spongiform encephalopathies in non-domestic animals: origin, transmission and risk factors.

The transmissible spongiform encephalopathies (TSEs), scrapie and bovine spongiform encephalopathy (BSE), are serious diseases of domestic animals. Although not as significant in terms of numbers of animals affected or geographical distribution, TSEs also affect non-domestic animals. Transmissible mink encephalopathy (TME) was the first TSE to be identified in non-domestic animals. This disease of captive mink (Mustela vison) is very rare and is associated with exposure through feed contaminated by a TSE agent. The second TSE to be identified in non-domestic animals was chronic wasting disease (CWD) of deer and elk. This disease is not known to be associated with feedstuffs contaminated with the agent of CWD, but the natural route of exposure appears to be oral, possibly through direct interaction between animals or through environmental contamination. Over the last five years, the known distribution of CWD across North America has expanded, increasing concerns over the impact of this disease on populations of free-ranging cervids and the viability of game farming industries. Concurrent with the epidemic of BSE, a variety of non-domestic ruminants and felid species were also affected in the United Kingdom, presumably through exposure to the agent in contaminated feed. These examples illustrate that when non-domestic animals are held in captivity, they depend upon feeds supplied by their caretakers and may show degrees of susceptibility to infectious agents in feeds which vary from those of domestic species. Although humans have less influence over exposure of free-ranging species to infectious agents, monitoring these populations for diseases may be important for managing the health of these animals. It is important to institute or continue surveillance for an entire range of infectious diseases, including TSEs, in free-ranging and captive non-domestic species. Study of diseases in these species may provide important information about infectious agents of concern for domestic animals and humans.

Rendering practices and inactivation of transmissible spongiform encephalopathy agents.

The authors describe the historic form of rendering and provide details on present-day practice. Possible future directions for the rendering industry are considered. The role of rendered meat-and-bone meal (MBM) as a dietary supplement in propagating the United Kingdom (UK) epidemic of bovine spongiform encephalopathy (BSE) is discussed, together with the role of MBM in spreading BSE outside the UK. Evidence that customarily used rendering processes did not substantially inactivate the agents of BSE or scrapie is presented. In addition, the influence that the abandonment of solvent extraction (as an adjunct to rendering) in the UK might have had on BSE infectivity levels in MBM is discussed. The BSE-related safety of tallow and by-products of tallow are considered. Data that associate the BSE agent with a new variant form of Creutzfeldt-Jakob disease in humans, predominantly but not exclusively, in the UK, are also discussed.

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups.

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).

Stability of the prion protein-encoding (PRNP) gene in HeLa cells

To assess the risk of the de novo emergence of the agent of transmissible spongiform encephalopathies in cultured cells, we examined the stability of the prion protein-encoding (PRNP) gene in HeLa cells and in cultures contaminated with HeLa cells that have been passaged extensively for over 50 years. Various sub-lineages of HeLa cells showed that some contained a mixture of a truncated PRNP gene (R3–R4 deletion) and a full-length PRNP gene, while others were homozygous for the R3–R4 deletion. That finding suggests that the progenitor of several popular sub-lineages of HeLa must have lost part or all of chromosome 20 early in the history of HeLa cells. No mutations were found in the PRNP genes. We conclude that the spontaneous appearance of mutations leading to expression of abnormal prion proteins in continuously passaged heteroploid cell lines is unlikely to pose a substantial risk for the safe production of biologicals in such cells.

Les risques alimentaires liés aux agents transmissibles non conventionnels (ATNC)

Transmissible spongiform encephalopathy (TSE) agents also named prions are responsible for transmissible subacute spongiform encephalopathies in humans and animals. TSE agents are thought to be composed only of an abnormal isoform of a host-coded protein, the prion protein (PrP). In vivo, raise in infectivity titer is always associated with a proportional accumulation of PrP. Aminoacid sequences of PrP obtained from normal and infected brains are identical, and these two proteins differ only by their biochemical and biophysical behaviour: in particular, PrP from TSE-affected individuals resists to proteinase K digestion. PrP-c is the PrP found in normal individuals, PrP-res is the proteinase K resistant PrP identifiable in infected individuals. TSE agents are capable to cross the species barrier. In march 96, a new variant of Creutzfeldt-Jakob disease (vCJD) has been described in UK. This emerging disease is now recognized to be induced by the BSE agent. Protection of human health is ensured by systematic PrP-res screening of cattle at slaughterhouse and by the specified risk material ban in the human and animal food chain.

Subclinical scrapie infection in a resistant species: persistence, replication, and adaptation of infectivity during four passages.

Cross-species infection with transmissible spongiform encephalopathy agents may lead to subclinical infection and to adaptation of the infection to new species. This is of particular concern for the millions of people possibly exposed to bovine spongiform encephalopathy (BSE) by consumption of BSE-infected beef. Subclinical infection was studied by making 4 serial passages of hamster scrapie agent (263K) in mice. At each step, infectivity was followed by inoculation of hamsters and mice. Subclinical infection was demonstrated either by detection of abnormal protease-resistant prion protein (PrP-res) or in the absence of PrP-res by detection of infectivity. Replication and adaptation of hamster infectivity in mice was shown in year 2 after initial mouse passage. In third and fourth passages, dual-tropic, mouse-tropic, and hamster-tropic infectivity was found in different animals. In some cases infectivity similar to the original 263K hamster scrapie strain was found after 2 or 3 serial mouse passages totaling 1200-1550 days.

Assessing the efficacy of a ram-genotyping programme to reduce susceptibility to scrapie in Great Britain

Susceptibility to clinical scrapie is associated with polymorphisms in the PrP gene. The ‘ARR’ allele of this gene reduces susceptibility to clinical disease caused by all known strains of the transmissible spongiform encephalopathy (TSE) agent. The British government proposes to use a ram-genotyping scheme to breed genetic resistance to clinical scrapie into the national sheep population. We considered how best to target limited genotyping resources to achieve the maximum rate of genotype evolution. We created a metapopulation model of the British sheep industry, which includes the major pure-breeds of sheep and the cross-breeds produced by crossing these pure-bred animals. The main criterion for assessing the efficacy of different strategies was the time taken to increase the prevalence of the ARR allele in the slaughter-lamb population. Our model predicted that the most-effective strategy would be to target genotyping to those rams used for pure-breeding (i.e. mated with the same breed of ewe). This strategy was compared to two further strategies, in which the proportion of rams genotyped in each breed depended on the prevalence of the ARR/ARR genotype in that breed. A policy in which the proportion of animals genotyped is reduced as the ARR prevalence in that breed increases is efficient. The most-effective policy was targeting the hill sector in the early years and gradually switching to genotyping more terminal-sire and longwool rams as the resistance of the hill sector increases.

PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts.

PrP(C) is a glycosylphosphatidylinositol-anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrP(C) remains unknown, a conformational isoform called PrP(Sc) (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrP(C) to PrP(Sc) conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild-type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrP(C) in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol-anchored proteins, we found that PrP(C) is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol-proteins, PrP(C) associates with detergent-resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol-proteins, as well as in the conversion process of PrP(C) to PrP(Sc). In order to discriminate whether detergent-resistant microdomains have a direct role in PrP(Sc) conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent-resistant microdomain association on PrP(C) intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol-linked protein, our data exclude a classical role for detergent-resistant microdomains in the post-trans-Golgi network sorting and transport of PrP(C) to the plasma membrane.

Molecular Basis of Scrapie Strain Glycoform Variation

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.

Prion diseases: pathogenesis and public health concerns

Transmissible spongiform encephalopathy (TSE) agents or prions induce neurodegenerative fatal diseases in humans and in some mammalian species. Human TSEs include Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome, kuru and fatal familial insomnia. In animals, scrapie in sheep and goats, feline spongiform encephalopathy, transmissible mink encephalopathy, chronic wasting disease in wild ruminants, and bovine spongiform encephalopathy (BSE), which appeared in the UK in the mid-1980s [Wells, G.A.H. et al. (1987) Vet. Rec. 121, 419–420], belong to the TSE group. Prions have biological and physicochemical characteristics that differ significantly from those of other microorganisms; for example, they are resistant to inactivation processes that are effective against conventional viruses, including those that alter nucleic acid structure or function. Alternatively, infectivity is highly susceptible to procedures that modify protein conformation. Today, the exact nature of prions remains unknown even though it is likely that they consist of protein only. At the biochemical level, TSEs are characterised by the accumulation, within the central nervous system of the infected individual, of an abnormal isoform of a particular protein from the host, the prion protein [Prusiner, S.B. (1982) Science 216, 136–144]. TSEs are transmissible among their species of origin, but they can also cross the species barrier and induce chronic infection and/or disease in other species. Transmissibility has been proven in natural situations such as the outbreak of CJD among patients treated with pituitary-derived hormones and the appearance of BSE that affected UK cattle in the mid-1980s.

Emerging infectious agents: do they pose a risk to the safety of transfused blood and blood products?

The blood supply is safer than it has been at any other time in recent history, and, in the context of other health care-related adverse events, the risks associated with blood transfusion are extremely small. The current high level of safety is the result of successive refinements and improvements in how blood is collected, tested, processed, and transfused; nonetheless, blood and plasma products remain vulnerable to newly identified or reemerging infections. In recent years, numerous infectious agents-including several newly discovered hepatitis viruses, the agents of transmissible spongiform encephalopathies, and tickborne pathogens-have been identified as potential threats to the safety of blood and plasma. Continued vigilance is critical to protect the blood supply from known pathogens and to monitor for the emergence of new infectious agents. Recent terrorist activities in the United States add new considerations to maintaining the safety and supply of blood. Education of clinicians and patients regarding the benefits and risks associated with the judicious use of blood and blood products can assist in informed decision making.

Characterization of Thermodynamic Diversity between Transmissible Spongiform Encephalopathy Agent Strains and Its Theoretical Implications

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes.

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6--7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells.

Migrating intestinal dendritic cells transport PrP(Sc) from the gut.

Bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease (vCJD) and possibly also sheep scrapie are orally acquired transmissible spongiform encephalopathies (TSEs). TSE agents usually replicate in lymphoid tissues before they spread into the central nervous system. In mouse TSE models PrP(c)-expressing follicular dendritic cells (FDCs) resident in lymphoid germinal centres are essential for replication, and in their absence neuroinvasion is impaired. Disease-associated forms of PrP (PrP(Sc)), a biochemical marker for TSE infection, also accumulate on FDCs in the lymphoid tissues of patients with vCJD and sheep with natural scrapie. TSE transport mechanisms between gut lumen and germinal centres are unknown. Migratory bone marrow-derived dendritic cells (DCs), entering the intestinal wall from blood, sample antigens from the gut lumen and carry them to mesenteric lymph nodes. Here we show that DCs acquire PrP(Sc) in vitro, and transport intestinally administered PrP(Sc) directly into lymphoid tissues in vivo. These studies suggest that DCs are a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases. To cite this article: H. Laude et al., C. R. Biologies 325 (2002) 49–57.

Involvement of macrophages in the pathogenesis of transmissible spongiform encephalopathies.

Although transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders, the immune system is also involved, at least in the early stages of their pathogenesis. Extensive studies have focused on cells targeted by the TSE agent for its replication but few on the possible involvement of macrophages in its clearance, as in more conventional diseases. This review summarises some of the experiments aimed at demonstrating a role for macrophages in TSE and presents the application to TSE of the macrophage "suicide" technique, which has been used to clarify the implication of these cells in the early steps of TSE pathogenesis.

Glycosylation influences cross-species formation of protease-resistant prion protein.

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease-resistant prion protein, PrP-res, from a normally soluble, protease-sensitive and glycosylated precursor, PrP-sen. While amino acid sequence similarity between PrP-sen and PrP-res influences both PrP-res formation and cross-species transmission of infectivity, the influence of co- or post-translational modifications to PrP-sen is unknown. Here we report that, if PrP-sen and PrP-res are derived from different species, PrP-sen glycosylation can significantly affect PrP-res formation. Glycosylation affected PrP-res formation by influencing the amount of PrP-sen bound to PrP-res, while the amino acid sequence of PrP-sen influenced the amount of PrP-res generated in the post-binding conversion step. Our results show that in addition to amino acid sequence, co- or post-translational modifications to PrP-sen influence PrP-res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.

Safety of oleochemical products derived from beef tallow or bone fat regarding prions

Bovine bone fat and beef tallow are widely used as raw material for oleochemical processes. Due to high temperature and pressure these processes are supposed to have an excellent inactivation potential for prions, i.e. the agent of BSE and other transmissible spongiform encephalopathies. To experimentally prove the destruction of the pathogenic prion protein aggregates under technically relevant conditions, oleochemical processes were emulated in the laboratory in reduced scale. Fat samples were spiked with highly infectious ex vivo prion rods and aliquots were taken before and after the processes. From these aliquots, undegraded prion protein was precipitated and determined by a sensitive Western blot assay. Degradation factors of 2 ?103 - 104 for catalytic fat hydrogenation and 107 for fat hydrolysis represent acceptable safety limits. With these experimental prion protein degradation factors the risk of human exposure to oleochemical products of bovine origin can be assessed. Assuming worst-case scenarios, the annual risk for human consumption of hydrogenated beef tallow is less than 6.8 x 10-7 and for skin application of fatty acid derivatives is less than 7.0 x 10-10. Both values are smaller than the background risk of contracting sporadic Creutzfeldt-Jakob disease (1 x 10-6 per annum).

Transmissible Spongiform Encephalopathy and Medicinal Products in the United States and Europe

Transmissible spongiform encephalopathy (TSE) is an inclusive term for a group of slowly progressing, neurodegenerative diseases with long incubation periods. Design and validation of manufacturing processes that eliminate TSE agents is difficult because of TSE resistance to standard chemical and physical measures and the lack of quick, reliable diagnostic assays. Thus, preventive measures have been put into place by regulatory authorities. Bovine spongiform encephalopathy (BSE), the cause of “mad cow disease,” is of particular concern since it is believed to cause Creutzfeld-Jakob disease in humans. Many medicinal products contain or come in contact with substances of bovine origin during manufacture. As a consequence, regulations and guidances have been established in the United States and Europe for bovine-derived materials used in manufacturing medicinal products. Manufacturers must assess active substances, excipients, raw materials, production reagents, and materials coming into direct contact with ...

Plasminogen binds to disease-associated prion protein of multiple species

The protein-only hypothesis states that the causative agent of transmissible spongiform encephalopathies is PrPSc, a conformer of the cellular protein PrPc. Therefore, reagents differentiating between PrPc and PrPSc could be diagnostically useful. Plasminogen, when immobilised onto magnetic beads, selectively precipitates PrPSc from mice with prion infected brains. We have shown that human plasminogen also precipitates PrPSc from brain homogenate of patients with sporadic Creutzfeldt-Jakob disease, as well as from sheep with scrapie and cows of various breeds with bovine spongiform encephalophathy (BSE). Our findings suggest that the binding of plasminogen to PrPSc could have diagnostic application. The protein-only hypothesis states that the causative agent of transmissible spongiform encephalopathies is PrPSc, a conformer of the cellular protein PrPc. Therefore, reagents differentiating between PrPc and PrPSc could be diagnostically useful. Plasminogen, when immobilised onto magnetic beads, selectively precipitates PrPSc from mice with prion infected brains. We have shown that human plasminogen also precipitates PrPSc from brain homogenate of patients with sporadic Creutzfeldt-Jakob disease, as well as from sheep with scrapie and cows of various breeds with bovine spongiform encephalophathy (BSE). Our findings suggest that the binding of plasminogen to PrPSc could have diagnostic application.