Spongiform Encephalopathy Agent Research Articles

Experimental Bovine Spongiform Encephalopathy in Squirrel Monkeys: The Same Complex Proteinopathy Appearing after Very Different Incubation Times.

Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a “species barrier,” often observed when TSE infections are transmitted from one species to another, would be expected to increase incubation periods compared with transmissions of same infectious agents within the same species. As part of a long-term study investigating the susceptibility to BSE of cell cultures used to produce vaccines, we inoculated squirrel monkeys (Saimiri sp., here designated SQ) with serial dilutions of a bovine brain suspension containing the BSE agent and monitored them for as long as ten years. Previously, we showed that SQ infected with the original “classical” BSE agent (SQ-BSE) developed a neurological disease resembling that seen in humans with variant CJD (vCJD). Here, we report the final characterization of the SQ-BSE model. We observed an unexpectedly marked difference in incubation times between two animals inoculated with the same dilution and volume of the same C-BSE bovine brain extract on the same day. SQ-BSE developed, in addition to spongiform changes and astrogliosis typical of TSEs, a complex proteinopathy with severe accumulations of protease-resistant prion protein (PrPTSE), hyperphosphorylated tau (p-tau), ubiquitin, and α-synuclein, but without any amyloid plaques or β-amyloid protein (Aβ) typical of Alzheimer’s disease. These results suggest that PrPTSE enhanced the accumulation of several key proteins characteristically seen in human neurodegenerative diseases. The marked variation in incubation periods in the same experimental TSE should be taken into account when modeling the epidemiology of human TSEs.

Infectivity-associated PrPSc and disease duration-associated PrPSc of mouse BSE prions

ABSTRACTDisease-related prion protein (PrPSc), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrPSc in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrPSc in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrPSc was precipitated into the pellet, and the supernatant contained only a slight amount of PrPSc. Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrPSc may be responsible for prion infectivity and that large, aggregated PrPSc may contribute to determining prion disease duration.

Hazardous Effect Free From Decontamination

Adventitious agents are those that are not inherent in the production of biopharmaceuticals. Microbial adventitious agents, Microbiocidal agents, help for inactivation of viruses, bacteria, fungi, mycoplasma, bacterial spore and sometimes prion. Bovine spongiform encephalopathy (BSE) agents, abnormal prions, are also potential adventitious agents. Adventitious agents can be introduced during establishment of cell lines, cell culture/fermentation, capture and downstream processing steps, formulation/filling, and even during drug delivery Therapeutic biotechnology products must have excellent safety characteristics. However, the potential introduction of adventitious agents must continually be evaluated for safety. The testing procedures that is performed for this purpose is addressed in regulatory documents that include ICH guidelines, European U.S., and Japanese Pharmacopoeia (EP,USP and JP) documents. In some cases, 9 CFR (U.S. Code of Federal Regulations) and 21 CFR, 211 and 610 are also applicable.

Laboratory activities involving transmissible spongiform encephalopathy causing agents

Since the appearance in 1986 of epidemic of bovine spongiform encephalopathy (BSE), a new form of neurological disease in cattle which also affected human beings, many diagnostic and research activities have been performed to develop detection and therapeutic tools. A lot of progress was made in better identifying, understanding and controlling the spread of the disease by appropriate monitoring and control programs in European countries. This paper reviews the recent knowledge on pathogenesis, transmission and persistence outside the host of prion, the causative agent of transmissible spongiform encephalopathies (TSE) in mammals with a particular focus on risk (re)assessment and management of biosafety measures to be implemented in diagnostic and research laboratories in Belgium. Also, in response to the need of an increasing number of European diagnostic laboratories stopping TSE diagnosis due to a decreasing number of TSE cases reported in the last years, decontamination procedures and a protocol for decommissioning TSE diagnostic laboratories is proposed.

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.

Stimulating the bovine spongiform encephalopathy situation in Japan

Despite various measures taken by the Japanese government to protect the cattle population from exposure to the bovine spongiform encephalopathy (BSE) agent, the first case of BSE was detected in September 2001. Subsequently, BSE surveillance was enhanced, involving mandatory reporting and investigation of all clinical BSE suspects, and testing of fallen stock and all cattle slaughtered for human consumption. Tests on over nine million cattle led to the detection of 35 additional cases by the end of May 2009. Using the surveillance data and other information as input variables, models were developed to explore the possible source of introduction of BSE into Japan, evaluate the effectiveness of control measures, estimate the prevalence of BSE in different birth cohorts, predict a future BSE epidemic, and simulate the impact of changes in surveillance strategies. Despite difficulties associated with the availability and uncertainty of some of the input variables, these models provided an objective insight into the BSE situation in Japan.

Replication and spread of CJD, kuru and scrapie agents in vivo and in cell culture

Transmissible Spongiform Encephalopathy (TSE) agents are defined by their virulence for particular species, their spread in the population, their incubation time to cause disease, and their neuropathological sequelae. Murine adapted human agents, including sporadic CJD (sCJD), New Guinea kuru, and Japanese CJD agents, display particularly distinct incubation times and maximal infectious brain titers. They also induce agent-specific patterns of neurodegeneration. When these TSE agents are transmitted to cultured hypothalamic GT1 cells they maintain their unique identities. Nevertheless, the human kuru (kCJD) and Japanese FU-CJD agents, as well as the sheep 22L and 263K scrapie agents display doubling times that are 8x to 33x faster in cells than in brain, indicating release from complex innate immune responses. These data are most consistent with a foreign viral structure, rather than an infectious form of host prion protein (PrP-res). Profound agent-specific inhibitory effects are also apparent in GT1 cells, and maximal titer plateau in kCJD and FU-CJD differed by 1,000-fold in a cell-based assay. Remarkably, the lower titer kCJD agent rapidly induced de novo PrP-res in GT1 cells, whereas the high titer FU-CJD agent replicated silently for multiple passages. Although PrP-res is often considered to be toxic, PrP-res instead may be part of a primal defense and/or clearance mechanism against TSE environmental agents. Limited spread of particular TSE agents through nanotubes and cell-to-cell contacts probably underlies the long peripheral phase of human CJD.

Is RNA critical for the production of TSE infection?

Is RNA critical for the production of TSE infection?

Microarray Analysis of Differentially Expressed Genes from Peyer's Patches of Cattle Orally Challenged with Bovine Spongiform Encephalopathy

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyer's patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

Fibrillization of recombinant bovine prion protein (rec-PrP) in vitro

112 Conversion of normal prion protein ( PrP c ) from the conformation with predominantly α -helical structure into the conformation rich in β -structures ( PrP sc , β -PrP, and PrP res ) underlies the pathogenesis of prion diseases, a special class of degenerative diseases of the nervous system of humans and animals. Prion diseases, or transmissible spongiform encephalopathies (TSEs) of humans, include the Creutzfeldt–Jakob disease (CJD), the Gerstmann–Straussler–Scheinker syndrome, new variant of CJD, kuru, as well as the fatal familial insomnia. The most well-known transmissible spongiform encephalopathies of animals are scrapie of sheep, chronic wasting disease of deer and moose, and bovine spongiform encephalopathy (mad cow disease) [1]. In the course of development of transmissible spongiform encephalopathies, amyloid fibrils (the so-called scrapie-associated fibrils) are formed in brain tissue of both animals and humans. These fibrils consist of β -PrP and are partially resistant to proteolysis. Transmissible spongiform encephalopathies differ in clinical symptoms, neuropathology, neuroanatomical distribution of PrP sc in tissues, and incubation period. It is believed that this heterogeneity of transmissible spongiform encephalopathies is determined by distinctions in the trimeric structure of pathological isoforms of PrP sc [1]. To understand the molecular mechanisms of conversion, the causes of strain differences of causative agents of transmissible spongiform encephalopathies, and interspecies transmission of prion diseases, transformation of recombinant PrP (rec-PrP) of human and different animal species into amyloid fibrils has been performed in vitro in the past years in several laboratories [3–6]. It was shown that the amino acid sequence of PrP markedly affects the formation and morphology of fibrils.

Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

Prion protein (PrP), the causative agent of Transmissible Spongiform Encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation, and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation.

Bovine Spongiform Encephalopathy Agent in a Prion Protein (PrP)ARR/ARRGenotype Sheep after Peripheral Challenge: Complete Immunohistochemical Analysis of Disease‐Associated PrP and Transmission Studies to Ovine‐Transgenic Mice

Possible transmission of the bovine spongiform encephalopathy (BSE) agent to ovine species has been considered for several years. It has been recently demonstrated that the BSE agent, after intracerebral challenge, can infect sheep believed to be the most resistant genetically to prion diseases (prion protein [PrP](ARR/ARR) genotype). We report here the results of a detailed immunohistochemical analysis of the disease-associated PrP (PrP(d)) in all organs from a PrP(ARR/ARR) sheep infected with the BSE agent by a peripheral route. Because PrP(d) was detected in the brain in the absence of any clinical symptoms, transmission studies were also performed using a sensitive ovine-transgenic mouse model--Tg(OvPrP4)--that can identify the BSE agent on the basis of the occurrence of florid plaques in the mouse brain. The data indicated that these PrP(d) deposits were linked to the BSE agent and were associated with infectivity. This suggests that PrP(ARR/ARR) sheep may be silent carriers of the BSE agent.

Polyanion induced fibril growth enables the development of a reproducible assay in solution for the screening of fibril interfering compounds, and the investigation of the prion nucleation site

The misfolded conformer of the prion protein (PrP) that aggregates into fibrils is believed to be the pathogenic agent in transmissible spongiform encephalopathies. In order to find fibril interfering compounds a screening assay in solution would be the preferred format to approximate more closely to physical conditions and enable the performance of kinetic studies. However, such an assay is hampered by the high irreproducibility because of the stochastic nature of the fibril formation process. According to published fibril models, the fibrillar core may be composed of stacked parallel β-strands. In these models positive charge repulsion may reduce the chance of favorable stacking and cause the irreproducibility in the fibril formation. This study shows that the charge compensation by polyanions induced a very strong fibril growth which made it possible to develop a highly reproducible fibril interference assay. The stimulating effect of the polyanions depended on the presence of the basic residues Lys106, Lys110 and His111. The assay was validated by comparison of the 50% fibril inhibition levels of peptide huPrP106-126 by six tetracyclic compounds. With this new assay, the fibrillogenic core (GAAAAGAVVG) of peptide huPrP106-126 was determined and for the first time it was possible to test the inhibition potentials of peptide analogues. Also it was found that variants of peptide huPrP106-126 with proline substitutions at positions Ala115, Ala120, or Val122 inhibited the fibril formation of huPrP106-126.

262 TISSUE-SPECIFIC ANALYSIS OF PRION EXPRESSION IN EARLY BOVINE FETUSES

The prion protein (PrP) is best known for its mis-folded, pathogenic isoform, which is widely regarded as the infectious agent in transmissable spongiform encephalopathies. However, the role of normal, cellular PrP, a host-encoded 29-kD glycoprotein tethered to the cell membrane by a phosphatidyl-inositol glycane (GPI) anchor, is poorly understood. PrP binds copper with high affinity, has antioxidant activity and may play a role in cell adhesion and/or signaling. PrP is expressed in mouse embryos on 6.5 days post-coitum in extra-embryonic tissue and at 13.5 days in the central and peripheral nervous system, intestine, and dental lamina. Our previous data revealed PrP gene expression in bovine embryos throughout pre-implantation embryo development. As part of a larger effort to map the ontogeny of cellular PrP expression in cattle, we sought here to analyze in early bovine fetuses (1) total PrP gene expression by real-time quantitative PCR (QPCR), and (2) tissue-specific PrP expression by immunohistochemistry. Fetuses were obtained from donor cattle bred by artificial insemination (AI; Day 0) and subjected to mid-ventral laparotomy on Days 32 (n = 2) and 39 (n = 2). Immediately upon recovery, one fetus from each stage was placed in RNAlater for RNA isolation and the other fixed in 10% formalin for immunohistochemistry. RNA was isolated using an RNeasy� mini kit (Qiagen, Valencia, CA, USA). cDNA was generated by reverse transcription with random hexamer priming and used the ΔΔcT method for estimation of PrP expression by QPCR. Tissue-specific expression was determined by immunohistochemistry. Formalin-fixed fetuses were embedded in paraffin, sagittally sectioned, dehydrated, and subjected to an unmasking protocol that employed Vectorlab unmasking solution and autoclaving. Tissues were then probed with a primary anti-PrP monoclonal antibody (SAF 32; Cayman Chemical Company, Ann Arbor, MI, USA). Bound primary antibody was detected with a biotinylated horse anti-mouse secondary antibody complexed to horseradish peroxidase using the ABC kit (Vector Laboratories, Burlingame, CA, USA). Probed sections were then counterstained with hematoxolin and eosin. Neighboring sections, processed identically but to which no primary antibody was added, served as controls. PrP gene expression was detected by QPCR at both stages examined and tended to be higher in Day 39 compared to Day 32 fetuses. PrP immunoreactivity was found throughout the central and peripheral nervous systems, ganglia, nerve trunks, and neural cell populations of sensory organs in both Day 32 and Day 39 fetuses. PrP immunolabeling was also observed in the mesonephric kidney, liver, and heart in the Day 39 fetus. At both stages, immunoreactivity was most intense in the nervous system. Thus, PrP is expressed in a tissue-specific pattern in early bovine fetuses. Tissue distribution of fetal PrP expression appears similar to that of adult PrP. Moreover, PrP appears to be expressed in a developmentally regulated fashion in some tissues.

The epidemiology of variant Creutzfeldt-Jakob disease.

Variant Creutzfeldt-Jakob disease (vCJD) was identified as a new disease in 1996. It was linked to infection with the bovine spongiform encephalopathy (BSE) agent although the epidemiological evidence for this was not strong, but later strain typing studies confirmed the association. The disease has affected predominantly young adults whose dietary and other characteristics are unexceptional compared to control groups, other than that all patients to date have been methoinine homozygous at codon 129 of the prion protein gene and the incidence has been about two times higher in the North of the UK. The number of cases in the 7 years after first identification of the disease has been considerably lower than initially feared, given the likely widespread exposure of the UK population to the BSE agent through contaminated beef products. Predictions of the possible future course of the epidemic have many associated uncertainties, but current mathematical models suggest that more than a few thousand cases is unlikely. Such modelling is limited by the absence of a test for infection with the vCJD agent. The development of a test that could be used on easily accessible tissue to detect infection early in the incubation period would not only advance understanding of the epidemiology of infection with the agent but would also aid the implementation of control measures to prevent potential iatrogenic spread.

Physiological and pathological functions of the prion protein homologue Dpl.

A misfolded version of the prion protein PrP(C), known as PrP(Sc), is the major component of scrapie infectivity, the pathological agent in transmissible spongiform encephalopathies. The Prnp gene that encodes the cellular PrP(C) protein was cloned almost 20 years ago, but remained without sequence or structural relatives for over a decade. Only recently a novel protein, named Doppel (Dpl), was identified, which shares significant biochemical and structural homology with PrP(C). When overexpressed, Dpl is neurotoxic and causes a neurological disease. Strikingly, Dpl neurotoxicity is counteracted and prevented by PrP(C). In contrast to its homologue PrP(C), Dpl is dispensable for prion disease progression and for the generation of PrP(Sc), but Dpl appears to have an essential function in male spermatogenesis. Although Dpl research is still in its infancy, the discovery of Dpl has already solved some enigmas of prion biology and an understanding of its physiological function is emerging.

The new variant of Creutzfeldt-Jakob disease.

New variant Creutzfeldt-Jakob disease (nvCJD) is a novel human transmissible spongiform encephalopathy which was first identified in 1996 in the United Kingdom (UK). Subsequent scientific studies have revealed that the strain of the transmissible agent responsible for nvCJD is identical to that of the bovine spongiform encephalopathy (BSE) agent, and the disease has been considered as 'human BSE'. By 31 December 1999, 52 cases of nvCJD had been reported (49 cases in the UK, two cases in France and one case in the Republic of Ireland). All these individuals were under 53 years of age and all those tested were methionine homozygotes at codon 129 of the prion protein gene. The number of cases of nvCJD likely to occur in the future is impossible to estimate because of multiple uncertainties, in particular the disease incubation period, the degree of exposure to the infective agent and the susceptibility of other genetic subtypes. Continued surveillance of both BSE and CJD is required in the UK and in other countries, to ensure that the scale of this potential epidemic is adequately monitored and that all possible steps are taken to prevent further human exposure to the BSE agent.

Bistability and the species barrier in prion diseases: stepping across the threshold or not

The infectious agent of transmissible spongiform encephalopathies is thought to be a cellular protein, the prion protein, which undergoes, under some circumstances, a dramatic conformational change leading to pathogenesis. The conversion between the normal and pathogenic isoforms corresponds to a autocatalytic mechanism and the metabolism of the prion protein exhibits switches between a normal, stable steady state and a pathogenic one. When the disease can be transmitted between two species, a primary infection from a heterologous donor has to be followed by two passages in the same host species so that the incubation period is stabilized. Sometimes, no pathogenic isoform of the prion protein is detected after the first passage, although corresponding brain extracts remain infectious. The observation that three and only three passages are needed in order to stabilize the strain strongly suggests that, during the course of the primary infection by the heterologous donor, an intermediary conformational species is formed. Within this assumption, a common mechanism involving only conformational changes of the prion protein can give a unifying interpretation of the problem of species barrier, lag characteristics and apparent lack of detection of the pathogenic isoform after the first passage in experiments dealing with interspecies transmission of prion diseases.

Second passage of a US scrapie agent in cattle

Scrapie and bovine spongiform encephalopathy are similar chronic neurodegenerative diseases of sheep and cattle. An earlier study showed that, on first passage in cattle, a US scrapie agent caused an encephalopathy that was distinct from bovine spongiform encephalopathy (BSE). The present report describes a second passage in cattle, carried out because diseases caused by the spongiform encephalopathy agents often change in character with additional passages in abnormal hosts. For this work, young calves were inoculated intracerebrally with a pooled suspension of brain from cattle that had died of encephalopathy after experimental inoculation with brain from scrapie-affected sheep. The second passage disease was essentially identical with the first passage disease, as judged by clinical signs, histopathological findings and distribution of "prion protein scrapie" (PrPsc). This represents additional evidence to suggest that the US sheep scrapie agent tested is incapable of causing BSE in cattle.

BSE Sets Agenda for Imported Gelatin

THE FALLOUT from "mad cow disease" continues, although incidence of the disease itself has been decreasing steadily since January 1993 (<i>JAMA</i>. 1996;276;438-440). The latest worry is a renewal of the uncertainties surrounding the risk of human exposure to the agent or agents of spongiform encephalopathy in gelatin, which is manufactured from the bones and hides of cows and pigs. The concern has prompted a Food and Drug Administration (FDA) committee to advise the agency to reimpose a restriction on the use of gelatin manufactured from parts of cows imported from countries that have reported cases of bovine spongiform encephalopathy (BSE). Humans are exposed to a wide variety of products that contain or use gelatin, from sausage casings and jellies to medicinal capsules and at least 1 vaccine. In January, for example, Italian health authorities suspended distribution of a<i>Haemophilus influenzae</i>b conjugate vaccine (named HibTITER, manufactured by Wyeth-Lederle Vaccines and