RESUMEN
Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy or prion disease affecting cervids. In 2016, the first cases of CWD were reported in Europe in Norwegian wild reindeer and moose. The origin and zoonotic potential of these new prion isolates remain unknown. In this study to investigate zoonotic potential we inoculated brain tissue from CWD-infected Norwegian reindeer and moose into transgenic mice overexpressing human prion protein. After prolonged postinoculation survival periods no evidence for prion transmission was seen, suggesting that the zoonotic potential of these isolates is low.
Asunto(s)
Ciervos , Priones , Reno , Enfermedad Debilitante Crónica , Animales , Ciervos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Noruega , Priones/genética , Priones/metabolismo , Reno/metabolismo , Enfermedad Debilitante Crónica/genéticaRESUMEN
The large chronic wasting disease (CWD)-affected cervid population in the USA and Canada, and the risk of the disease being transmitted to humans through intermediate species, is a highly worrying issue that is still poorly understood. In this case, recombinant protein misfolding cyclic amplification was used to determine, in vitro, the relevance of each individual amino acid on cross-species prion transmission. Others and we have found that the ß2-α2 loop is a key modulator of transmission barriers between species and markedly influences infection by sheep scrapie, bovine spongiform encephalopathy (BSE), or elk CWD. Amino acids that differentiate ovine and deer normal host prion protein (PrPC) and associated with structural rigidity of the loop ß2-α2 (S173N, N177T) appear to confer resistance to some prion diseases. However, addition of methionine at codon 208 together with the previously described rigid loop substitutions seems to hide a key in this species barrier, as it makes sheep recombinant prion protein highly susceptible to CWD-induced misfolding. These studies indicate that interspecies prion transmission is not only governed just by the ß2-α2 loop amino acid sequence but also by its interactions with the α3-helix as shown by substitution I208M. Transmissible spongiform encephalopathies, characterized by long incubation periods and spongiform changes associated with neuronal loss in the brain, have been described in several mammalian species appearing either naturally (scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, Creutzfeldt-Jakob disease in humans) or by experimental transmission studies (scrapie in mice and hamsters). Much of the pathogenesis of the prion diseases has been determined in the last 40 years, such as the etiological agent or the fact that prions occur as different strains that show distinct biological and physicochemical properties. However, there are many unanswered questions regarding the strain phenomenon and interspecies transmissibility. To assess the risk of interspecies transmission between scrapie and chronic wasting disease, an in vitro prion propagation method has been used. This technique allows to predict the amino acids preventing the transmission between sheep and deer prion diseases.
Asunto(s)
Ciervos/metabolismo , Proteínas Priónicas/metabolismo , Ovinos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Pollos , Ratones Noqueados , Proteínas Priónicas/química , Dominios Proteicos , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad de la EspecieRESUMEN
This is the first volume of the Handbook of Clinical Neurology totally devoted to prion diseases. The reason for this choice is to inform neurologists and neuroscientists about the remarkable advances that this field has made in the diagnosis of human and animal prion diseases, understanding the pathogenesis of disease, and in the development of novel in vivo and in vitro models. In recent years, the knowledge of prion replication and mechanisms of prion spreading within the brain and peripheral organs of infected people has also become important for understanding other protein misfolded diseases of the brain, such as Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis. Researchers in these diseases have recognized that the process within an individual leading to the deposition of misfolded proteins within the central nervous system shares remarkable common mechanisms with prion diseases, leading to the terminology of "prion-like diseases."
Asunto(s)
Enfermedades por Prión/etiología , Enfermedades por Prión/metabolismo , Priones/metabolismo , Pliegue de Proteína , Animales , HumanosRESUMEN
The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), causes variant Creutzfeldt-Jakob disease (vCJD) in humans following dietary exposure. While it is assumed that all cases of vCJD attributed to a dietary aetiology are related to cattle BSE, sheep and goats are susceptible to experimental oral challenge with cattle BSE prions and farmed animals in the UK were undoubtedly exposed to BSE-contaminated meat and bone meal during the late 1980s and early 1990s. Although no natural field cases of sheep BSE have been identified, it cannot be excluded that some BSE-infected sheep might have entered the European human food chain. Evaluation of the zoonotic potential of sheep BSE prions has been addressed by examining the transmission properties of experimental brain isolates in transgenic mice that express human prion protein, however to-date there have been relatively few studies. Here we report that serial passage of experimental sheep BSE prions in transgenic mice expressing human prion protein with methionine at residue 129 produces the vCJD phenotype that mirrors that seen when the same mice are challenged with vCJD prions from patient brain. These findings are congruent with those reported previously by another laboratory, and thereby strongly reinforce the view that sheep BSE prions could have acted as a causal agent of vCJD within Europe.
Asunto(s)
Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob , Proteínas Priónicas/metabolismo , Priones/metabolismo , Factores de Edad , Animales , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patología , Síndrome de Creutzfeldt-Jakob/transmisión , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Transgénicos , Fenotipo , Proteínas Priónicas/genética , OvinosRESUMEN
Spiroplasma spp., tiny filterable wall-less bacteria, are consistently associated with the transmissible spongiform encephalopathies (TSE). Spiral forms have been transiently isolated from TSE-affected brain tissues in SP4 growth media designed for isolation of Spiroplasma spp., but the isolate could not be propagated in SP4 media. A bacterium must grow in vitro in cell-free cultures to allow full characterization of a suspect pathogen. Here, a novel Spiroplasma sp. was isolated from scrapie- and chronic wasting disease (CWD)-affected brains and lymph nodes. Filtrates of tissue homogenates inoculated into Brucella media incubated for 14 days at 35 °C resulted in high titers of spiroplasma as shown by dark-field microscopy. A drop assay of infected media on Bacto Schaedler agar showed spiroplasma isolates forming unique subsurface colonies after 21 days incubation. Spiroplasma coils, coccoid forms and clumps of entwined spiroplasma filaments were seen on the agar by scanning electron microscopy. Since Brucella media has a sodium bisulfite additive that lowers oxygen tension, TSE spiroplasma growth requires media with low oxygen tension. Brucella media allows for isolation and propagation of spiroplasma from TSE-affected tissues, which will lead to complete characterization of this TSE pathogen and determine its role as a candidate causative agent of TSE.
Asunto(s)
Encéfalo/microbiología , Ganglios Linfáticos/microbiología , Enfermedades por Prión/microbiología , Spiroplasma/aislamiento & purificación , Animales , Encéfalo/patología , Enfermedades por Prión/patología , OvinosRESUMEN
Transmissible spongiform encephalopathies (TSE) or prion diseases exhibit strain variation, a phenomenon that has been studied extensively in mouse bioassays. Despite the introduction of many rapid in vitro systems, bioassays remain a key tool in defining prion strains and their ability to transmit disease in vivo. Prion strains can be characterized by a range of phenotypic characteristics such as incubation period, vacuolar pathology, and distribution of the abnormal form of PrP following experimental transmission of the agent into a panel of mice (transgenic or wild type). Interpretation of these characteristics requires considerable experience and an understanding of the procedures used to define them. This chapter reviews the techniques used in strain typing of prion diseases from inoculum preparation and pathological studies to data interpretation alongside an extensive troubleshooting guide.
Asunto(s)
Bioensayo , Encéfalo/patología , Proteínas PrPC/química , Proteínas PrPSc/química , Enfermedades por Prión/patología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Marcha , Expresión Génica , Histocitoquímica/métodos , Ratones , Ratones Transgénicos , Microtomía/métodos , Fenotipo , Proteínas PrPC/clasificación , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/clasificación , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Especificidad de la Especie , Adhesión del Tejido/métodos , Fijación del Tejido/métodosRESUMEN
Across the spectrum of sporadic human prion diseases (also known as transmissible spongiform encephalopathies: TSE), there is considerable phenotypic diversity. Cumulative scientific evidence supports that prions, the infectious agents of prion diseases, are constituted predominantly, if not exclusively, by misfolded, typically protease-resistant, disease-associated isoforms of the prion protein (PrPres). Consequently, tissue deposition of PrPres is considered a hallmark of prion disease pathology, and this can be visualized by Western blotting after tissue homogenization and treatment with proteinases, particularly proteinase K (PK). Indeed, Western blot profiles of PrPres are utilized as one marker of different prion strains, with such strains thought to contribute to at least part of the phenotypic variation observed in sporadic human prion disease. Typically, Western blotting of PrPres demonstrates three bands of different electrophoretic mobility, depicting the di-glycosylated, mono-glycosylated and unglycosylated species although further subclassification and the delineation of novel sporadic disease subtypes, such as variably protease-sensitive prionopathy, has contributed greater complexity. Nevertheless, it is the mobility of the unglycosylated PrPres band, the relative abundance of the two glycosylated bands or overall profile of the banding post-PK, in combination with the prion protein gene (PRNP) codon 129 genotype that allows the categorisation of molecular subtypes of sporadic human prion disease. These subtypes appear to correlate with distinct clinico-pathological profiles of sporadic Creutzfeldt-Jakob disease.
Asunto(s)
Western Blotting/métodos , Síndrome de Creutzfeldt-Jakob/diagnóstico , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas PrPC/química , Proteínas Priónicas/clasificación , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Endopeptidasa K/química , Expresión Génica , Glicosilación , Humanos , Fenotipo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Pliegue de ProteínaRESUMEN
In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrPTSE) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrPTSE, measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrPTSE seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.
Asunto(s)
Encefalopatía Espongiforme Bovina/prevención & control , Heparina/aislamiento & purificación , Proteínas Priónicas/aislamiento & purificación , Animales , Bovinos , Encefalopatía Espongiforme Bovina/transmisión , Intestinos/química , Mesocricetus , Scrapie/transmisión , Hidróxido de Sodio , Extractos de TejidosRESUMEN
Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal neurodegenerative disorders affecting several mammalian species. Its causative agent, disease-associated prion protein (PrPd), is a self-propagating ß-sheet rich aberrant conformation of the cellular prion protein (PrPC) with neurotoxic and aggregation-prone properties, capable of inducing misfolding of PrPC molecules. PrPd is the major constituent of prions and, most importantly, is the first known example of a protein with infectious attributes. It has been suggested that similar molecular mechanisms could be shared by other proteins implicated in diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis or systemic amyloidoses. Accordingly, several terms have been proposed to collectively group all these disorders. Through the stringent evaluation of those aspects that characterise TSE-causing prions, in particular propagation and spread, strain variability or transmissibility, we will discuss whether terms such as "prion", "prion-like", "prionoid" or "propagon" can be used when referring to the aetiological agents of the above other disorders. Moreover, it will also be discussed whether the term "infectious", which defines a prion essential trait, is currently misused when referring to the other misfolded proteins.
Asunto(s)
Enfermedades por Prión/metabolismo , Enfermedad de Alzheimer/metabolismo , Amiloidosis/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Humanos , Enfermedad de Parkinson/metabolismo , Pliegue de ProteínaRESUMEN
Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.