Scrapie infection and endogenous retroviral expression in sheep lymphoid tissues.

Transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative diseases affecting humans and animals. Although many host tissues express PrPC (essential for prion replication), relatively few cell types accumulate significant levels of infectivity, including neurons and other cell types in the nervous system, and follicular dendritic cells in secondary lymphoid organs. This suggests that tissue or cell-specific receptors or cofactors could play a role in controlling differential susceptibility to infection. Endogenous retroviruses (ERV), the remnants of ancient retroviral integration into the host germline, may represent one such cofactor. We examined the effect of scrapie infection on expression of three ovine ERV families (enJSRV/ß1-OERV, γ1-OERV, γ2-OERV) in secondary lymphoid tissues of sheep at different time points following subcutaneous inoculation, using RT-qPCR. These OERVs were constitutively expressed in the prescapular lymph node and spleen of uninfected sheep. However, we were unable to find convincing evidence of specific differential expression of OERV in the same tissues following scrapie infection, in contrast to previous studies of ERV expression in brains of prion-infected mice and macaques. This study is the first to quantify the expression of potentially functional OERV transcripts in sheep lymphoid tissues, opening up interesting questions about the consequences for host immune function.

Scrapie diagnosis in a goat and four Santa Inês sheep from the same herd in Brazil / Diagnóstico de scrapie em um caprino e quatro ovinos Santa Inês de um mesmo rebanho no Brasil

Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Inês sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm...

Scrapie é uma encefalopatia espongiforme transmissível (EET) progressiva e fatal de ocorrência natural em ovinos e caprinos. A suspeita de scrapie é baseada nos sinais clínicos, porém a manifestação patológica da proteína priônica (PrP) nos tecidos-alvo é necessária para a confirmação da doença. A presença de uma forma anormal da proteína (PrPSc) em tecido linforreticular e tecido nervoso constitui uma característica importante para o diagnóstico. Este trabalho é o relato de um foco de scrapie ocorrido em rebanho com 55 ovinos mistos Suffolk, 21 caprinos e 19 ovinos Santa Inês, na região Centro-Oeste do Brasil. Os animais foram eutanasiados após a confirmação de um caso de scrapie com sinais clínicos em um ovino Suffolk nessa propriedade...

Perspectives of a scrapie resistance breeding scheme targeting Q211, S146 and K222 caprine PRNP alleles in Greek goats.

The present study investigates the potential use of the scrapie-protective Q211 S146 and K222 caprine PRNP alleles as targets for selective breeding in Greek goats. Genotyping data from a high number of healthy goats with special emphasis on bucks, revealed high frequencies of these alleles, while the estimated probabilities of disease occurrence in animals carrying these alleles were low, suggesting that they can be used for selection. Greek goats represent one of the largest populations in Europe. Thus, the considerations presented here are an example of the expected effect of such a scheme on scrapie occurrence and on stakeholders.

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

The effects of host age on the transport of complement-bound complexes to the spleen and the pathogenesis of intravenous scrapie infection.

Infections with variant Creutzfeldt-Jakob disease (vCJD) have almost exclusively occurred in young patients, but the reasons for this age distribution are uncertain. Our data suggest that the pathogenesis of many peripherally acquired transmissible spongiform encephalopathy (TSE) agents is less efficient in aged individuals. Four vCJD cases linked to transfusion of vCJD-contaminated blood or blood products have been described. Three cases occurred in elderly patients, implying that intravenous exposure is more efficient in aged individuals than other peripheral routes. To test this hypothesis, young (6 to 8 weeks old) and aged (600 days old) mice were injected intravenously with a TSE agent. In aged and young mice, the intravenous route was more efficient than other peripheral routes of TSE agent exposure. However, in aged mice, disease pathogenesis was significantly reduced. Although most aged mice failed to develop clinical disease during their life spans, many showed histopathological signs of TSE disease in their brains. Thus, the effects of age on intravenous TSE pathogenesis may lead to significant levels of subclinical disease in the population. After peripheral exposure, many TSE agents accumulate upon follicular dendritic cells (FDCs) in lymphoid tissues before they infect the brain. In aged spleens, PrP(C) expression and TSE agent accumulation upon FDCs were reduced. Furthermore, the splenic marginal zone microarchitecture was substantially disturbed, adversely affecting the delivery of immune complexes to FDCs. This study is the first to suggest that the effects of aging on the microarchitecture and the function of the splenic marginal zone significantly influence the pathogenesis of an important pathogen.

Molecular characterization of the full-length coding sequence of the caprine laminin receptor gene (RPSA).

Scrapie is a prion disease in sheep and goats. Ribosomal protein SA (RPSA), also called 37 kDa laminin receptor precursor/67 kDa laminin receptor has been demonstrated to be a putative cell surface receptor for prion. To investigate the caprine RPSA, we cloned the full-length coding sequence of the gene of goat and submitted it to GenBank. The length of the open reading frame is 888 bp, encoding 295 amino acids. The putative amino acid sequence is highly similar to that of other mammals. The caprine amino acid sequence of RPSA is shown to be identical to the sequence of species susceptible to scrapie at positions 241, 272, and 291. The phylogenetic tree analysis revealed that the genetic distance between sheep and goat is the smallest. Moreover, RT-PCR results of 11 tissues indicated that RPSA mRNA is expressed in all selected caprine tissues.

Elimination capacity of a TSE-model agent in the manufacturing process of Alphanate/Fanhdi, a human factor VIII/VWF complex concentrate.

The variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate) and Fanhdi in their ability to eliminate an experimental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into samples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromatography and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a laboratory scale model representative of the industrial manufacturing process. The prion protein (PrP(Sc)) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the production process: 3.21-3.43 log(10) for the PEG precipitation; about 3.45 log(10) for the affinity chromatography; and around 2.0 log(10) for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demonstrated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log(10) can be deduced. The tests from the production process of FVIII/VWF complex concentrates have demonstrated their potential for eliminating TSE agents.

Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock.

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.

Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.

Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles.

BACKGROUND INFORMATION: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a). RESULTS: The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs. CONCLUSIONS: The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

Sodium valproate does not augment Prpsc in murine neuroblastoma cells.

Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 microM VPA. Higher VPA concentrations (240 and 600 microM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.

Disease-specific particles without prion protein in prion diseases - phenomenon or epiphenomenon?

The search for the cause of transmissible spongiform encephalopathies (TSEs) has a long and tortuous history. In a recent paper, 25-nm virus-like particles were identified that were consistently observed in cell cultures infected with Creutzfeldt-Jakob disease (CJD) and scrapie; they are similar to, or even identical with, the virus-like tubulovesicular structures (TVS) found in experimental scrapie as early as in 1968, and subsequently in all naturally occurring and experimentally induced TSEs. These particles have been viewed with caution by the scientific community because of the unverified or uninterpretable record of virus-like structures reported over the years in TSEs. TVS are spherical or tubular particles of approximate diameter 25-37 nm. They are smaller than synaptic vesicles, but larger than many particulate structures of the central nervous system, such as glycogen granules. Their electron density is higher compared with synaptic vesicles, and in experimental murine scrapie, they form paracrystalline arrays. None of these observations distinguish between TVS as an entity critical to the infectious process, or as a highly specific ultrastructural epiphenomenon, but their consistent presence in all TSEs demands further research.

Cells infected with scrapie and Creutzfeldt-Jakob disease agents produce intracellular 25-nm virus-like particles.

We had repeatedly found approximately 25-nm-diameter virus-like particles in highly infectious brain fractions with little prion protein (PrP), and therefore we searched for similar virus-like particles in situ in infected cell lines with high titers. Neuroblastoma cells infected with the 22L strain of scrapie as well as hypothalamic GT cells infected with the FU Creutzfeldt-Jakob disease agent, but not parallel mock controls, displayed dense 25-nm virus-like particles in orthogonal arrays. These particles had no relation to abnormal PrP amyloid in situ, nor were they labeled by PrP antibodies that faithfully recognized rough endoplasmic reticulum membranes and amyloid fibrils, the predicted sites of normal and pathological intracellular PrP. Additionally, phorbol ester stimulated the production of abnormal PrP gel bands by >5-fold in infected N2a + 22L cells, yet this did not increase either the number of virus-like arrays or the infectious titer of these cells. Thus, the 25-nm infection-associated particles could not be prions. Synaptic differentiation and neurodegeneration, as well as retroviruses that populate the rough endoplasmic reticulum of neuroblastoma cells, were not required for particle production. The 25-nm particle arrays in cultured cells strongly resembled those first described in 1968 in synaptic regions of scrapie-infected brain and subsequently identified in many natural and experimental TSEs. The high infectivity of comparable, isolated virus-like particles that show no intrinsic PrP by antibody labeling, combined with their loss of infectivity when nucleic acid-protein complexes are disrupted, make it likely that these 25-nm particles are the causal TSE virions that induce late-stage PrP brain pathology.

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: implication of MuLV in progression of scrapie pathogenesis.

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.

Subclinical scrapie infection in a resistant species: persistence, replication, and adaptation of infectivity during four passages.

Cross-species infection with transmissible spongiform encephalopathy agents may lead to subclinical infection and to adaptation of the infection to new species. This is of particular concern for the millions of people possibly exposed to bovine spongiform encephalopathy (BSE) by consumption of BSE-infected beef. Subclinical infection was studied by making 4 serial passages of hamster scrapie agent (263K) in mice. At each step, infectivity was followed by inoculation of hamsters and mice. Subclinical infection was demonstrated either by detection of abnormal protease-resistant prion protein (PrP-res) or in the absence of PrP-res by detection of infectivity. Replication and adaptation of hamster infectivity in mice was shown in year 2 after initial mouse passage. In third and fourth passages, dual-tropic, mouse-tropic, and hamster-tropic infectivity was found in different animals. In some cases infectivity similar to the original 263K hamster scrapie strain was found after 2 or 3 serial mouse passages totaling 1200-1550 days.

Removal of prion challenge from an immune globulin preparation by use of a size-exclusion filter.

BACKGROUND: A size-exclusion filter (Viresolve 180, Millipore Corp.) was tested for its ability to remove transmissible spongiform encephalopathies prion protein from an immune globulin preparation during ultrafiltration. STUDY DESIGN AND METHODS: Hamster-adapted 263K scrapie brain homogenate (SBH) was spiked into Rh0(D) immune globulin (human) at 1 in 300 and 1 in 1000 dilutions. Before spiking, the SBH was treated with detergent, sonicated, and filtered through serial 0.45-, 0.22-, and 0.1-microm filters to present a rigorous filter challenge. Process variables were monitored throughout the ultrafiltration to ensure that the spiked material did not compromise the membrane flux. Removal of scrapie prion protein (PrP(Sc)) material was determined by use of a sensitive Western blot assay. RESULTS: The turbid SBH became completely translucent after sonication and passage through the 0.45-, 0.22-, and 0.1-microm filters. The filtration of the immune globulin containing PrP(Sc) material was more difficult to perform than was filtration of immune globulin spiked with the normal cellular isoform. Even during tangential flow filtration, the fibril material prevented the PrP(Sc)-spiked immune globulin from passing as readily through the filter. Western blot results indicated a removal of greater than or equal to 2.5 log PrP(Sc), while remaining within the normal filtration limits. CONCLUSIONS: The composition, physical condition, and the amount of SBH introduced have significant effects on the filtration of the immune globulin and the log removal values obtained. By use of a detergent-treated, sonicated, and filtered preparation of SBH, it was demonstrated that the Viresolve 180 effectively removes PrP(Sc) from the immune globulin.

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene.

We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.

Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent.

A sensitive immunohistochemical procedure was used to investigate the presence of prion protein (PrP) in the ileal Peyer's patch of PrP-genotyped lambs, including scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. The tyramide signal amplification system was used to enhance the sensitivity of conventional immunohistochemical procedures to show that PrP was widely distributed in the enteric nervous plexus supplying the gut wall. In scrapie-free lambs, PrP was also detected in scattered cells in the lamina propria and in the dome and interfollicular areas of the Peyer's patch. In the follicles, staining for PrP was mainly confined to the capsule and cells associated with vascular structures in the light central zone. In lambs naturally exposed to the scrapie agent, staining was prominent in the dome and neck region of the follicles and was also found to be associated with the follicle-associated epithelium. Similar observations were made in lambs that had received a single oral dose of scrapie-infected brain material from sheep with a homologous and heterologous PrP genotype 1 and 5 weeks previously. These studies show that the ileal Peyer's patch in young sheep may be an important site of uptake of the scrapie agent and that the biology of this major gut-associated lymphoid tissue may influence the susceptibility to oral infection in sheep. Furthermore, these studies suggest that homology or heterology between PrP genotypes or the presence of PrP genotypes seldom associated with disease does not impede uptake of PrP.