Oral transmissibility of prion disease is enhanced by binding to soil particles

Soil may serve as an environmental reservoir for prion infectivity and contribute to the horizontal transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) of sheep, deer, and elk. TSE infectivity can persist in soil for years, and we previously demonstrated that the disease-associated form of the prion protein binds to soil particles and prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. Here, we assess the oral infectivity of Mte- and soil-bound prions. We establish that prions bound to Mte are orally bioavailable, and that, unexpectedly, binding to Mte significantly enhances disease penetrance and reduces the incubation period relative to unbound agent. Cox proportional hazards modeling revealed that across the doses of TSE agent tested, Mte increased the effective infectious titer by a factor of 680 relative to unbound agent. Oral exposure to Mte-associated prions led to TSE development in experimental animals even at doses too low to produce clinical symptoms in the absence of the mineral. We tested the oral infectivity of prions bound to three whole soils differing in texture, mineralogy, and organic carbon content and found soil-bound prions to be orally infectious. Two of the three soils increased oral transmission of disease, and the infectivity of agent bound to the third organic carbon-rich soil was equivalent to that of unbound agent. Enhanced transmissibility of soil-bound prions may explain the environmental spread of some TSEs despite the presumably low levels shed into the environment. Association of prions with inorganic microparticles represents a novel means by which their oral transmission is enhanced relative to unbound agent.     

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.     

Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.     

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than ～15 nm in diameter).     

A simple, versatile and sensitive cell-based assay for prions from various species

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.     

White Blood Cell-Based Detection of Asymptomatic Scrapie Infection by Ex Vivo Assays

Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods - in vitro, ex vivo and in vivo assays - to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.     

Potential role of soil properties in the spread of CWD in western Canada

Chronic wasting disease (CWD) is a horizontally transmissible prion disease of free ranging deer, elk and moose. Recent experimental transmission studies indicate caribou are also susceptible to the disease. CWD is present in southeast Alberta and southern Saskatchewan. This CWD-endemic region is expanding, threatening Manitoba and areas of northern Alberta and Saskatchewan, home to caribou. Soil can serve as a stable reservoir for infectious prion proteins; prions bound to soil particles remain infectious in the soils for many years. Soils of western Canada are very diverse and the ability of CWD prions to bind different soils and the impact of this interaction on infectivity is not known. In general, clay-rich soils may bind prions avidly and enhance their infectivity comparable to pure clay mineral montmorillonite. Organic components of soils are also diverse and not well characterized, yet can impact prion-soil interaction. Other important contributing factors include soil pH, composition of soil solution and amount of metals (metal oxides). In this review, properties of soils of the CWD-endemic region in western Canada with its surrounding terrestrial environment are described and used to predict bioavailability and, thus, potential spread of CWD. The major soils in the CWD-endemic region of Alberta and Saskatchewan are Chernozems, present in 60% of the total area; they are generally similar in texture, clay mineralogy and soil organic matter content, and can be characterized as clay loamy, montmorillonite (smectite) soils with 6–10% organic carbon. The greatest risk of CWD spread in western Canada relates to clay loamy, montmorillonite soils with humus horizon. Such soils are predominant in the southern region of Alberta, Saskatchewan and Manitoba, but are less common in northern regions of the provinces where quartz-illite sandy soils with low amount of humus prevail.     

Accelerated shedding of prions following damage to the olfactory epithelium

In this study, we investigated the role of damage to the nasal mucosa in the shedding of prions into nasal samples as a pathway for prion transmission. Here, we demonstrate that prions can replicate to high levels in the olfactory sensory epithelium (OSE) in hamsters and that induction of apoptosis in olfactory receptor neurons (ORNs) in the OSE resulted in sloughing off of the OSE from nasal turbinates into the lumen of the nasal airway. In the absence of nasotoxic treatment, olfactory marker protein (OMP), which is specific for ORNs, was not detected in nasal lavage samples. However, after nasotoxic treatment that leads to apoptosis of ORNs, both OMP and prion proteins were present in nasal lavage samples. The cellular debris that was released from the OSE into the lumen of the nasal airway was positive for both OMP and the disease-specific isoform of the prion protein, PrP(Sc). By using the real-time quaking-induced conversion assay to quantify prions, a 100- to 1,000-fold increase in prion seeding activity was observed in nasal lavage samples following nasotoxic treatment. Since neurons replicate prions to higher levels than other cell types and ORNs are the most environmentally exposed neurons, we propose that an increase in ORN apoptosis or damage to the nasal mucosa in a host with a preexisting prion infection of the OSE could lead to a substantial increase in the release of prion infectivity into nasal samples. This mechanism of prion shedding from the olfactory mucosa could contribute to prion transmission.     

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic'' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.     

Resistance of soil-bound prions to rumen digestion

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrP(c) from complete digestion, while both unbound and soil-bound infectious PrP(Sc) proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment.     

Photodegradation illuminates the role of polyanions in prion infectivity

Understanding the mechanism by which prion infectivity is encoded by the misfolded protein PrP^Sc remains a high priority within the prion field. Work from several groups has indicated cellular cofactors may be necessary to form infectious prions in vitro. The identity of endogenous prion conversion cofactors is currently unknown, but may include polyanions and/or lipid molecules. In a recent study, we manufactured infectious hamster prions containing purified PrP^Sc, co-purified lipid, and a synthetic photocleavable polyanion. The polyanion was incorporated into infectious PrP^Sc complexes, and then specifically degraded by exposure to ultraviolet light. Light-induced in situ degradation of the incorporated polyanion had no effect on the specific infectivity of the samples as determined by end-point dilution sPMCA and scrapie incubation time assays. Furthermore, prion strain properties were not changed by polyanion degradation, suggesting that intact polyanions are not required to maintain the infectious properties of hamster prions. Here, we review these results and discuss the potential roles cofactors might play in encoding prion infectivity and/or strain properties.     

Scrapie Agent (Strain 263K) can transmit disease via the oral route after persistence in soil over years

The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrP(Sc) and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrP(Sc) in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.     

Elucidating the role of cofactors in mammalian prion propagation

Perhaps the most intriguing scientific question about mammalian prions is how these proteinaceous entities encode and propagate infectivity. Over the past decade, our laboratory has taken a reductionist biochemical approach to study this challenging question. Our studies have resulted in the identification of endogenous phospholipid and polyanionic cofactor molecules that facilitate prion formation in vitro. Using these cofactor molecules, we have been able to produce purified, chemically defined prions with high levels of specific infectivity for wild type animal hosts. Our most recent studies suggest that cofactor molecules may also play crucial roles in maintaining the infectious conformation and strain properties of mammalian prions. The ability to produce high titer prions in vitro using cofactors provides an unprecedented opportunity to study the structural mechanism of infectious prion formation directly.     

Prions adhere to soil minerals and remain infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrP(Sc)) with common soil minerals. In this study, we demonstrated substantial PrP(Sc) adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrP(Sc)-binding capacities of each mineral. Furthermore, we observed that PrP(Sc) desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrP(Sc) and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrP(Sc) bound to Mte remained infectious. Results from our study suggest that PrP(Sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.     

Organic polyanions act as complexants of prion protein in soil

The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.     

Dual nature of the infectious prion protein revealed by high pressure

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrP(Sc)) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 degrees C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrP(Sc) or the N-truncated PrP-(27-30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6-7 log(10) reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive beta-structures (PrP(Sc)(DeltaPsen)) and extremely pressure-resistant beta-structures (PrP(Sc)(DeltaPres)). Remarkably, after the first centrifugation step at 540,000 x g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrP(Sc) under pressure, suggesting a kind of semicrystalline polymer structure.     

Efficient Lymphoreticular Prion Propagation Requires PrPc in Stromal and Hematopoietic Cells

In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrP(c)). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrP(c) (Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrP(c) on hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation of Prnp in either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrP(c) expression by both stromal and hematopoietic compartments.     

Prion Stability and Infectivity in the Environment

The biology of normal prion protein and the property of infectivity observed in abnormal folding conformations remain thinly characterized. However, enough is known to understand that prion proteins stretch traditional views of proteins in biological systems. Numerous investigators are resolving details of the novel mechanism of infectivity, which appears to feature a protein-only, homologous replication of misfolded isoforms. Many other features of prion biology are equally extraordinary. This review focuses on the status of infectious prions in various natural and man-made environments. The picture that emerges is that prion proteins are durable under extreme conditions of environmental exposure that are uncommon in biological phenomena, and this durability offers the potential for environmental reservoirs of persistent infectivity lasting for years. A recurrent theme in prion research is a propensity for these proteins to bind to mineral and metal surfaces, and several investigators have provided evidence that the normal cellular functions of prion protein may include metalloprotein interactions. This structural propensity for binding to mineral and metal ions offers the hypothesis that prion polypeptides are intrinsically predisposed to non-physiological folding conformations that would account for their environmental durability and persistent infectivity. Similarly, the avidity of binding and potency of prion infectivity from environmental sources also offers a recent hypothesis that prion polypeptides bound to soil minerals are actually more infectious than studies with purified polypeptides would predict. Since certain of the prion diseases have a history of epidemics in economically important animal species and have the potential to transmit to humans, urgency is attached to understanding the environmental transmission of prion diseases and the development of protocols for their containment and inactivation. Special issue article in honor of Dr. George DeVries.     

Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤10(1)- to ≥10(5.5)-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants.