The protein component of scrapie-associated fibrils is a glycosylated low molecular weight protein.

Scrapie-associated fibril protein (SAF-protein) extracted from infectious scrapie-associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF-protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N-linked carbohydrate chains represent approximately 40-60% of the mol. wt. of SAF-protein. The deglycosylated SAF-protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF-protein is an amyloid-type of protein. The simplest explanation for the available data is that SAF-polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host-encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co-purification of scrapie virus and SAF caused by the high carbohydrate content of SAF-protein.     

Persistence of infectious HIV on follicular dendritic cells

Follicular dendritic cells (FDCs) trap Ags and retain them in their native state for many months. Shortly after infection, HIV particles are trapped on FDCs and can be observed until the follicular network is destroyed. We sought to determine whether FDCs could maintain trapped virus in an infectious state for long periods of time. Because virus replication would replenish the HIV reservoir and thus falsely prolong recovery of infectious virus, we used a nonpermissive murine model to examine maintenance of HIV infectivity in vivo. We also examined human FDCs in vitro to determine whether they could maintain HIV infectivity. FDC-trapped virus remained infectious in vivo at all time points examined over a 9-mo period. Remarkably, as few as 100 FDCs were sufficient to transmit infection throughout the 9-mo period. Human FDCs maintained HIV infectivity for at least 25 days in vitro, whereas virus without FDCs lost infectivity after only a few days. These data indicate that HIV retained on FDCs can be long lived even in the absence of viral replication and suggest that FDCs stabilize and protect HIV, thus providing a long-term reservoir of infectious virus. These trapped stores of HIV may be replenished with replicating virus that persists even under highly active antiretroviral therapy and would likely be capable of causing infection on cessation of drug therapy.     

Infectivity of the DNA from four isolates of JC virus.

The infectivity of JC virus DNA was demonstrated in its most permissive cell culture, primary human fetal glial cells. The amount of infectivity observed in these heterogeneous cultures varied considerably between batches of cells. Contrary to results obtained with the papovaviruses simian virus 40 and BK virus, the calcium technique (F. L. Graham and A. J. van der Eb, Virology 52:456--467, 1973) was found to be more efficient at promoting JC virus DNA infectivity than the DEAE-dextran method (J. H. McCutchan and J. S. Pagano, J. Natl. Cancer Inst. 41:351--357, 1968): maximum infectivity titers of 4 x 10-(4) and 6 x 10(3) fluorescent cell units per microgram of DNA, respectively. These values represent an approximate recovery of infectivity from virus of between 0.02 and 0.14%. Comparisons of infectivity of DNAs obtained from four isolates of JC virus and which differed in their degrees of heterogeneity did not reveal significant differences. The JC virus DNA was not infectious in primary human fetal lung and kidney cells.     

Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans

Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.     

Retrovirus infection strongly enhances scrapie infectivity release in cell culture

Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.     

Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.     

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

It has been suggested that the infectious agents of scrapie and Creutzfeldt-Jakob disease (CJD) are 'prions' constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re-evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase-K-resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one-half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.     

The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities

The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action.     

Scrapie infectivity in hamster blood is not associated with platelets

The infectivity of hamster scrapie strain 263K was measured in platelets isolated from blood pooled from six hamsters with clinical scrapie. The total number of infectious doses present in the blood pool was 220, out of which only 3.5 infectious doses were associated with platelets. A larger proportion of the total infectivity was recovered from the mononuclear leukocyte fraction. This result indicates that platelets are not the source of blood-borne infectivity in transmissible spongiform encephalopathy-infected hamsters.     

High incidence of scrapie induced by repeated injections of subinfectious prion doses

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.     

Isolation and characterization of a succinimide variant of methionyl human growth hormone.

Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Monoclonal antibodies which preferentially bind to 22 K human growth hormone rather than its 20 K variant

We have established 13 hybridoma cell lines which secrete mouse IgG1 monoclonal antibodies (McAbs) to human growth hormone (hGH). Binding affinity and binding specificity of McAbs were analyzed by competitive radioimmunoassay. Among these McAbs, CL. B1 showed a high affinity of 9.8 x 10(8) l/mol, and all McAbs so far tested showed very weak cross-reactivity or none at all with human prolactin (hPRL) and human chorionic somatomammotropin (hCS; human placental lactogen). Analysis of binding sites of McAbs using hGH variant and fragments in both ELISA and RIA demonstrated that McAbs could be classified into two groups. All the McAbs obtained in this study bound to plasmin-digested fragment S2 (hGH 1-134 and 141-191) and fragment alpha 3 (hGH 1-134 and 147-191). However, five (such as 1D2) out of 13 McAbs bound to fragment F1 (hGH 1-134) and others (such as CL. B1) did not. The McAb CL. B1 in the latter group showed low affinity with 20 K hGH (residue 32-46 deleted in native 22 K hGH) in contrast to high affinity with hGH (22 K). This suggests that the former McAbs recognize an epitope located at the N-terminal two-third part of hGH. In contrast, the McAbs of the latter group are likely to recognize three-dimensional structure of native 22 K hGH.     

Infectivity of scrapie prions bound to a stainless steel surface.

BACKGROUND: The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity. MATERIALS AND METHODS: As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease. RESULTS: Stainless steel wire (0.15 x 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 10(5) LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold. CONCLUSIONS: The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments.     

The infectivity and lytic activity of minute virus of mice wild-type and derived vector particles are strikingly different

Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.     

Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32)

Fcgamma receptor (FcgammaR)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. FcgammaRIA (CD64) and FcgammaRIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory gamma-chain subunit that associates with FcgammaRIA and constitutively in FcgammaRIIA is required for phagocytosis mediated by these receptors. To determine whether FcgammaRIA and FcgammaRIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that FcgammaRIIA appeared to do so far more effectively. Abrogation of FcgammaRIA signaling competency, either by expression without gamma-chain or by coexpression with gamma-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcgammaRIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between FcgammaRIA and FcgammaRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcgammaRs.     

Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.     

Equilibrium denaturation of human growth hormone and its cysteine-modified forms

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.     

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.