Longitudinal Detection of Prion Shedding in Saliva and Urine by Chronic Wasting Disease-Infected Deer by Real-Time Quaking-Induced Conversion.

UNLABELLED: Chronic wasting disease (CWD) is an emergent, rapidly spreading prion disease of cervids. Shedding of infectious prions in saliva and urine is thought to be an important factor in CWD transmission. To help to elucidate this issue, we applied an in vitro amplification assay to determine the onset, duration, and magnitude of prion shedding in longitudinally collected saliva and urine samples from CWD-exposed white-tailed deer. We detected prion shedding as early as 3 months after CWD exposure and sustained shedding throughout the disease course. We estimated that the 50% lethal dose (LD50) for cervidized transgenic mice would be contained in 1 ml of infected deer saliva or 10 ml of urine. Given the average course of infection and daily production of these body fluids, an infected deer would shed thousands of prion infectious doses over the course of CWD infection. The direct and indirect environmental impacts of this magnitude of prion shedding on cervid and noncervid species are surely significant. IMPORTANCE: Chronic wasting disease (CWD) is an emerging and uniformly fatal prion disease affecting free-ranging deer and elk and is now recognized in 22 U.S. states and 2 Canadian provinces. It is unique among prion diseases in that it is transmitted naturally through wild populations. A major hypothesis to explain CWD's florid spread is that prions are shed in excreta and transmitted via direct or indirect environmental contact. Here we use a rapid in vitro assay to show that infectious doses of CWD prions are in fact shed throughout the multiyear disease course in deer. This finding is an important advance in assessing the risks posed by shed CWD prions to animals as well as humans.

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates.

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.

In situ photodegradation of incorporated polyanion does not alter prion infectivity.

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrP(Sc) aggregates in vivo. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrP(Sc), we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrP(C) substrate, PC-oligo was incorporated into physical complexes with PrP(Sc) molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrP(Sc) molecules alone.

Expert elicitation for the judgment of prion disease risk uncertainties.

There is a high level of uncertainty surrounding the potential for iatrogenic prion transmission through transplantation, medical instrument reuse, blood transfusion, and blood product use due to a lack of evidence-based research on this important risk issue. A group of specialists was enlisted to evaluate some of the knowledge gaps in this area using the "Classical Model," a structured elicitation procedure for weighting and pooling expert judgment. The elicitation exercise was undertaken in March 2009 with 11 transmissible spongiform encephalopathy (TSE) experts who were first calibrated using a series of seed questions for which the answers are known; they were then asked to answer a number of target questions that are important for risk assessment purposes, but for which there remains high uncertainty at this time. The target questions focused on variant Creutzfeldt-Jakob disease (vCJD) prevalence, incubation times for vCJD, genetic susceptibility to prion disease, blood infectivity, prion reduction of blood and blood products, surgical instrument risks, and interspecies transmission of TSEs. The experts were also asked to perform pairwise risk rankings for 12 different potential routes of infection. Dura mater transplantation was seen as having the highest risk, while dental tissue grafts were viewed as presenting the lowest risk of iatrogenic transmission. The structured elicitation procedure provides a rational, auditable, and repeatable basis for obtaining useful information on prion disease risk issues, for which data are sparse.

Prions: who should worry about them?

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are a family of neurodegenerative disorders affecting both humans and animals. They are caused by the accumulation of an abnormal form of a protein known as prion that results in neuronal death and a characteristic spongiform appearance of the brain tissue. Human prion diseases can be sporadic, acquired or hereditary. Acquired prion diseases have been linked to entering contaminated food into the human food chain, failure to completely disinfect or sterilize contaminated surgical instruments, patients receiving tissues and organs from infected donors, recipients of blood and other biological contaminated products, and potentially to cross infection in dental procedures. At present, there is unfortunately no efficient therapy that can be administered to clinically infected patients with prion diseases. Moreover, there are no simple diagnostic tests that can be used to show the agent of transmissible spongiform encephalopathy during the preclinical phase of the disease. Therefore, to prevent the spread of this emerging infectious agent it is necessary to implement several health control strategies and maintain surveillance for subclinical infections.

Prion diseases and iatrogenic infections I. A review.

Iatrogenic diseases are disorders caused by the treatment of physician or surgeon (iatros from Greek "healer"). The disease develops by transmission of prion-infected material from the (clinically inapparent) donor to the recipient. First case significantly confirmed by animal bioassay was described in a corneal transplant 1974 and another following a neurosurgical procedure in 1977. Over one hundred of cases were detected in recipients of cadaveric, prion infected pituitary hormones (mostly growth hormone or, significantly less, gonadotrophins); the majority in France, UK and USA. Over one hundred of cases were detected following transplantation of prion-infected dura mater (mostly lyophilized commercial preparations) mostly in Japan. Some hormonal as well dura mater cases still occur because of an enormously prolonged incubation period. There are no fresh cases because cadaveric hormones were replaced by synthetic preparations and cadaveric dura mater by autologous tissue (fascia lata, fascia temporalis). Actual problems of iatrogenic prion infections are confined to surgery/neurosurgery, ophthalmology, otorhinolaryngology and dental surgery. Prions have also been detected outside the central nervous system, posterior eye, peripheral nerves, muscles, spleen, lymphoreticular system as tonsils and appendix, intestine, urine (?), olfactory cilia and central olfactory pathway representing a route of infection (nasal secretions). General anaesthesia may also be involved. Medical devices in contact with infected tissues became contaminated within minutes. Iatrogenic infections may occur thereafter incubating for years or decades. They are difficult to register because the hospital documentation has been actually kept for ten years only. It is evident that prion diseases have frequently a surgical history, according to some authors in one third of patients. Another problem of greatest importance is the prion decontamination of infected medical devices that is really difficult at present and rarely, if ever, properly performed in greatest majority of world hospitals. The decontamination methods will be presented in the second part of this article.

The hydrophobic internal region of bovine prion protein shares structural and functional properties with HIV type 1 fusion peptide.

The conserved fusion peptide at the N-terminus of HIV-1 envelope glycoprotein gp41 is involved in the virus-cell fusion reaction and in the cytopathic effects promoted by expression in single cells. The conserved bovine prion protein 121KHVAGAAAAGAVVGGLGGYMLGSAMSR147 transmembrane region (BPrP(tm)) contains a sequence rich in Gly residues [i.e., 130GAVVGGLGGYMLGSAMSR147 (BPrP(mi))] that shows homology with HIV-1 fusion peptide. As in the case of the latter peptide, analysis of the BPrP(mi) interfacial hydrophobicity confirms that this stretch bears an intrinsic tendency to partitioning from the aqueous phase into membranes. Experimental analyses of BPrP(mi)-lipid interactions suggest several similarities between this sequence and HIV-1 fusion peptide. Infrared spectroscopy reveals a conformational plasticity of the membrane-associated prion sequence comparable to that displayed by the viral sequence. The adoption of a mainly alpha-helical structure correlates with the formation of lytic pores. This helical structure can be converted into a beta-sheet conformation by addition of calcium, a process that is accompanied by vesicle membrane fusion. In contrast, transmembrane BPrP(tm) associates with membranes in a nonlytic, mainly helical structure but also containing some random coil. Upon addition of calcium, the random coils disappear while the helical conformation remains. In the absence of membranes both prion and HIV-1 sequences form amyloid-type fibers. It is proposed that during the pathological processes induced by secreted PrPSc and its proteolytic fragments, conformational polymorphism displayed by membrane-inserted BPrP(mi) may play a role at perturbing the general architecture of the membrane lipid bilayer and inducing protein-protein aggregation at membrane surfaces. These findings suggest the existence of common mechanisms underlying cytotoxicity by PrP and HIV-1 gp41.

Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies.

OBJECTIVES: To establish an animal model to study transmissible spongiform encephalopathy using hamsters and steel wires contaminated with infectious brain materials as transfer vehicles, and, based on this model, to test decontamination procedures against the infectious prion proteins on the steel wires as a near real situation bioassay. DESIGN: Infectious brain materials were given to healthy hamsters intracerebrally either as a suspension or as dried materials on the surface of steel wires. The animals were observed for 18 months. During this period, animals showing definitive clinical signs were euthanized. Decontamination studies were performed by reprocessing contaminated steel wires with different disinfection agents and procedures before implantation. RESULTS: Pathological prion proteins were able to bind to the steel wires and caused disease after the contaminated wires were implanted in the brains of hamsters. When the contaminated wires were treated with different reprocessing procedures before implantation, infectivity was reduced, which was manifested directly by prolonged survival time of the test animals. These results show that this model can be used as a bioassay to validate reprocessing procedures for surgical instruments. CONCLUSIONS: At the time of submission of this article, only the group of hamsters incubated with wires reprocessed with an alkaline detergent, followed by sterilization with a modified cycle in a hydrogen peroxide gas plasma sterilizer (4 injections), showed no clinical signs of disease and remained alive. Two animals from the group receiving sodium hydroxide followed by autoclaving (at 134 degrees C for 18 minutes) died. Furthermore, the tested enzymatic cleaning agent seemed to have no positive effect.

Prions and the oral cavity.

Prion diseases have recently emerged as a significant challenge to health-care workers, including those involved in dentistry. Abnormal prion proteins are resistant to complete inactivation by conventional sterilization techniques. In the last decade, a new form of prion disease emerged in the UK, termed "variant CJD", thought to be acquired by consumption of bovine spongiform encephalopathy-contaminated food products. At present, CJD is an invariably fatal disease with no immediate prospect of treatment or vaccination. Of concern with the variant form of CJD, unlike the more classic forms of the disease, is the appearance of significant levels of infectivity outside the central nervous system. This raises concerns for the potential transmission of prion proteins via surgical procedures from individuals in the asymptomatic stage of the disease. This article reviews the existing knowledge base on the nature of prions, their distribution in oral tissues, and the implications for dental treatment.

Prions and the human transmissible spongiform encephalopathies.

The last 5 years have seen the emergence of a new disease in humans (vCJD), mainly in the United Kingdom. This emergence has been accompanied by an explosion of scientific data on a novel group of the responsible infectious agents called prions and has profound implications for infection control and transfusion policies. Also of concern is the finding of prions in neural, gingival, pulpal, and salivary tissue in animal models and significant titers of infectivity from extraneural organs (particularly, in cases of vCJD, in lymphoreticular tissues). There is limited information on the presence of prion proteins in the oral tissues from human studies. Because of the differences in patterns of disease in animal models and in strains of prion protein, it is difficult to extrapolate directly these findings to humans, but it illustrates a potential for transmission by way of the dental route. High levels of infectivity may be present in tissues early in the incubation period and before clinical signs and symptoms. The dental profession must turn its attention to the routine decontamination of dental instruments to ensure that these procedures are performed to the highest regulatory standard. Clinicians and manufacturers must work closely together to develop instruments that are either single use or can be presented in a form that can be more easily decontaminated. Clinicians must pay close attention to manufacturers' decontamination instructions and must not reuse items designated as single use, such as endodontic files. Improvements in compliance with these requirements will not only reduce the risk of transmission of TSEs but also other less tenacious infectious agents.

Prion diseases: risks, characteristics, and infection control considerations in dentistry.

Prion diseases are a group of fatal neurodegenerative diseases that are rapidly progressive and fatal, with no definite cure. There are no reported cases of prion disease transmission arising from dental procedures. Nevertheless, there is a theoretical but real risk of transmission of prion disease from dental instruments. A review was made of studies up to 2008 to provide an update of the characteristics, risk of transmission, and the infection-control implications of prions in the field of dentistry. As the prions are resistant to conventional sterilization methods, highly-specific, cross-infection control measures are required when managing patients infected with these.

Enzymatic detergent treatment protocol that reduces protease-resistant prion protein load and infectivity from surgical-steel monofilaments contaminated with a human-derived prion strain.

The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extraneural tissue distribution of variant and sporadic Creutzfeldt-Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. In this study, the ability of preparations of enzymatic medical instrument cleaners to reduce the infectivity associated with a rodent-adapted strain of human prion disease, previously reported to be resistant to decontamination, was tested. Efficient degradation of the disease-associated prion protein by enzymatic cleaning preparations required high treatment temperatures (50-60 degrees C). Standard decontamination methods (1 M NaOH for 1 h or autoclaving at 134 degrees C for 18 min) reduced infectivity associated with the human-derived prion strain by less than 3 log10 LD50. In contrast, a 30 min treatment with the optimized enzymatic cleaning preparation protocols reduced infectivity by more than 3 log10 LD50 and when used in conjunction with autoclave cycles eliminated detectable levels of infectivity. The development of prion decontamination procedures that are compatible with routine cleaning and sterilization of medical and surgical instruments may reduce the risk of the transmission of prion disease in general surgery.

Cell-free formation of misfolded prion protein with authentic prion infectivity.

Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Properties of scrapie prion protein liposomes.

Purified scrapie prions contain one identifiable macromolecule, PrP 27-30, which polymerizes into rod-shaped amyloids. The rods can be dissociated with retention of scrapie infectivity upon incorporation of PrP 27-30 into detergent-lipid-protein complexes (DLPC) as well as liposomes. As measured by end-point titration, scrapie infectivity was increased greater than 100-fold upon dissociating the rods into liposomes. The incorporation of PrP 27-30 into liposomes was demonstrated by immunoelectron microscopy using colloidal gold. Detergent extraction of prion liposomes followed by chloroform/methanol extraction resulted in the reappearance of rods, indicating that this process is reversible. Scrapie prion infectivity in rods and liposomes was equally resistant to inactivation by irradiation at 254 nm and was unaltered by exposure to nucleases. A variety of lipids used for producing DLPC and liposomes did not alter infectivity. Fluorescently labeled PrP 27-30 in liposomes was used to study its entry into cultured cells. Unlike the rods which remained as large fluorescent extracellular masses, the PrP 27-30 in liposomes rapidly entered the cells and was seen widely distributed within the interior of the cell. PrP 27-30 is derived by limited proteolysis from a larger protein designated PrP(Sc) which is membrane bound. PrP(Sc) in membrane fractions was solubilized by incorporation in DLPC, thus preventing its aggregation into amyloid rods. The functional solubilization of scrapie prion proteins in DLPC and liposomes offers new approaches to the study of prion structure and the mechanism by which they cause brain degeneration.

[About prions...]. / A propos des prions....

Iatrogenic Creutzfeldt-Jakob disease (CJD) was first reported in 1974, in a 55-year-old woman whose symptoms started 18 months following corneal implant surgery. The transplant donor died of CJD. More recently, the epidemy of bovine spongiform encephalopathy in the U.K., and the reported cases of iatrogenic CJD due to extractive pituitary hormone injections, emphasized the problems of its etiology and the way these neurodegenerative diseases get transmitted. A new infectious pathogen was described as a prion: "small proteinaceous infectious particle", responsible of transmissible neurodegenerative diseases. The lethal evolution of these diseases, and the complete absence of preventive procedures are fearful regarding the extension of the disease, specially during the procedure of grafting originating from possibly infected people whose screening is currently impossible. It is mandatory for the surgeon to update its knowledges including the legal bylaws regarding a good surgical prevention. One must be certain the implant, wether autologous or heterologous, is completely free of disease, mainly in aesthetic surgery. This paper attempts to summarise this topics. One must bear in mind that the current knowledges could soon turn obsolete with a constant progression of scientific research and of the epidemiologic data.

Infectivity of liposomally encapsulated nucleic acids isolated from EMC virus and scrapie-infected mouse brain.

The ability of liposomes to enhance nucleic acid infectivity in vivo was studied. Encephalomyocarditis (EMC) virus RNA was extracted and encapsulated within liposomes. The infectivity of EMC RNA was increased by liposomal entrapment after intraperitoneal injection of mice, but decreased after intracerebral injection. In contrast, when nucleic acids from scrapie-infected brains were entrapped in liposomes and injected into mice by one of four routes, no cases of scrapie were observed. This is the first report of the enhancement of nucleic acid infectivity by liposomes in vivo.

Purified prion proteins and scrapie infectivity copartition into liposomes.

Considerable evidence indicates that the scrapie prion protein (PrP 27-30) is required for infectivity. Aggregates of PrP 27-30 form insoluble amyloid rods that resist dissociation by nondenaturing detergents. Mixtures of the detergent cholate and phospholipids were found to solubilize purified PrP 27-30 in the form of detergent-lipid-protein complexes. Removal of the cholate by dialysis resulted in the formation of closed liposomes. Both the detergent-lipid-protein complexes and the liposomes often but not always exhibited a 10-fold increase in scrapie infectivity compared to that observed with the rods. No evidence for a prion-associated nucleic acid could be found when the phospholipid vesicles containing PrP 27-30 were digested with nucleases and Zn2+ under conditions that allowed hydrolysis of exogenously added nucleic acids. No filamentous or rod-shaped particles were found amongst prion liposomes by electron microscopy in our search for a putative filamentous "scrapie virus." The partitioning of PrP 27-30 and scrapie infectivity into phospholipid vesicles contends that PrP 27-30 has a central role in scrapie pathogenesis, establishes that the prion amyloid rods are not essential for infectivity, and argues that prions are fundamentally different from viruses.