Scrapie incubation periods and end-point titers in mouse strains differing at the H-2D locus

In extending findings on the influence of the mouse H-2D locus on the scrapie incubation period, we showed that with the intracerebral (i.c.) route of injection, SJL and NZW mice (s and z alleles, respectively) had shorter incubation periods than C57BL mice (b allele) at several concentrations of two scrapie strains, ME7 and 139A. The three mouse strains have the same Sinc genotype, s7s7. Incubation period data among the three mouse strains after intraperitoneal (i.p.) injection revealed a different rank order of incubation periods from that seen after i.c. injection. End-point titers in the three mouse strains were similar after i.c. injection, but both scrapie strains yielded very low titers in NZW mice after i.p. injection. There were marked differences between 139A and ME7 incubation periods after both i.c. and i.p. injections in the three mouse genotypes, providing an additional parameter that distinguishes these two scrapie strains.     

Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i.e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.     

Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene

Amino acid polymorphisms in the prion protein gene (PrP) affect the susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy (TSE). In particular, amino acid substitutions at codons 136, 154 and 171 of the ovine PrP gene are associated with different degrees of susceptibility to the classical form of scrapie, caused by 'typical' scrapie strains. Existing genotyping tests for scrapie susceptibility normally interrogate only the single nucleotide polymorphisms (SNPs) most relevant to 'typical' strains. Recently, however, a number of novel variants of the scrapie agent have been discovered. The ability of these new, 'atypical' scrapie variants to infect sheep that are resistant to 'typical' variants has raised concerns about the reduction in genetic variability that may result from intense selection for resistance to classical scrapie. Furthermore, a growing interest in a potential role for specific PrP genotypes in modulating performance traits is also driving a move toward more extensive characterization of haplotypes at the PrP locus. Here, we describe a single-tube method for the interrogation of eight SNPs within seven codons (112, 136, 141, 154, 171, 231 and 241) of the ovine PrP gene. This method is as accurate as sequencing, yet more affordable, and can easily be automated for high-throughput sample screening. Moreover, it can be modified to accommodate genetic variations that are found in local and heritage breeds.     

Profound sex-specific effects on incubation times for transmission of bovine spongiform encephalopathy to mice

Four strains of mice were inoculated intracerebrally with a primary isolate of bovine spongiform encephalopathy (BSE) and the cloned mouse-adapted scrapie strain ME7. Clinical prion disease diagnosis was made at the appearance of three or more neurological symptoms and their persistence for 3 consecutive weeks and confirmed by neuropathological criteria. For BSE, incubation periods were profoundly different between the sexes in all four mouse strains, being longer in the females. In contrast, ME7 scrapie incubation times were similar between the sexes. Our results indicate that sex-specific processes are involved in the course of primary BSE transmission. Research into this phenomenon may provide clues to the prophylaxis of BSE and have possible implications for new variant Creutzfeldt-Jakob disease in humans.     

Absence of autoantibodies against neurofilament proteins in the sera of scrapie infected mice

Autoantibodies against neurofilament proteins were not detected in any of the sera from the following scrapie infected mice: 19 mice infected with scrapie agent 139A in pre-clinical stage, 32 histologically confirmed scrapie mice and other 12 clinical scrapie mice infected with various strains. The test sera were assayed against acetone-fixed central neuron cultures from fetal mice by indirect immunofluorescence and immunoperoxidase techniques. The negative result suggests that autoantibodies against neurofilament proteins do not play a role in the pathogenesis of scrapie.     

Partial copurification of scrapie-associated fibrils and scrapie infectivity

The association between scrapie infectivity and scrapie-associated fibrils (SAF) during a partial purification procedure for infectivity was investigated. Scrapie infectivity and SAF can be separated from most membrane components by subcellular fractionation of infected mouse brain to obtain a synaptosomal fraction, followed by detergent treatment and density gradient centrifugation. After different detergent treatments, with either octyl glucoside or sodium N-lauroyl sarcosinate, SAF showed differing sedimentation characteristics but nevertheless cosedimented with scrapie infectivity in both cases. Copurification under two different conditions provides more evidence that SAF may be a form of the infectious agent of scrapie.     

Acceleration of scrapie disease in mice by an adenovirus

Coinfected mice were examined for a possible interaction between the scrapie agent and an adenovirus. A low titer (10(2) TCD50) of mouse adenovirus (MAdV) caused a significant acceleration of clinical signs of scrapie in mice infected 128 days previously with scrapie. In this experiment, the coinfected mice died 19 days earlier than mice infected with scrapie alone. When a higher titer of MAdV (10(4)-10(5) PFU) was used, a more drastic acceleration of scrapie disease was seen in mice infected 85 and 110 days previously with scrapie. At 85 days, coinfection caused mice to die 37 days earlier than mice infected with scrapie alone, whereas at 110 days, coinfection caused mice to die 52 days earlier than mice infected with scrapie alone. MAdV alone caused no clinical disease in normal mice. The brains of coinfected mice and mice that had been infected with scrapie alone showed a histopathology consistent with scrapie. A possible explanation for these findings is that the replication of the scrapie agent is accelerated by adenovirus. Defective parvoviruses are known to be helped by adenoviruses. Spleens from coinfected mice but not from mice infected with MAdV alone yielded, in cultures of BALB 3T3 cells, infectious MAdV and one or two smaller agents with the dimension and shape of a parvovirus.     

Disappearance of scrapie virus from tissues of the mouse

Examination of newborn mice, inoculated intraperitoneally with high doses of scrapie virus, revealed that the virus could not be reisolated from their tissues after about 1 week following inoculation, until almost 1 year later. The inoculum was rapidly removed and was not detectable, although the animals became latently infected. Homogenization of whole inoculated newborn animals showed that only about 3% of virus could be recovered by the 2nd day postinoculation (p.i.). During the first 6 days p.i. the half-life of titratable scrapie was about 15 h. A further study of the rate of disappearance of clarified scrapie virus from blood after intravenous inoculation showed an even more rapid disappearance, with a half-life of 5.16 min. Prior treatment of the recipient mice with either carbon black or silica to block the reticuloendothelial system (RES) did not affect the rate of disappearance. It was concluded that the mouse possesses a very efficient means of scrapie virus removal from the blood which is not dependent upon an active RES. However, after 1 h the rate of disappearance changed dramatically; the residual virus level was very stable, with no significant drop during the next 21 h. This finding was compatible with the possibility that two forms of scrapie virus, with different removal rates, coexisted in the inoculum. Silica treatment caused a shortened scrapie incubation period.     

Scrapie and chronic wasting disease

Scrapie and CWD share many features. There are marked similarities in the clinical presentations, the lesions, and the pathogenesis of these diseases, and some similarities in the epidemiology. Extrapolation from the scrapie model of TSE disease to CWD--which occurs in three different species, and should not be considered to be uniform in their response--may be erroneous, however. Such differences may influence diagnostics (e.g., the amount and distribution of PrPC in these different species), pathogenesis (e.g., the influence of genetics on susceptibility and resistance), and epidemiology (e.g., the mode and dynamics of transmission and influences of domestication). IHC is used widely for diagnostics and in the study of the pathogenesis of scrapie and CWD. This technique holds promise for antemortem diagnosis of infection in the peripheral lymphoid tissues such as lymphoid follicles of the nictitating membrane and the tonsil.     

Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.     

青藏高原岩羊朊蛋白基因及氨基酸序列分析

目的 分析岩羊的朊蛋白基因(PRNP)序列,评估岩羊对羊瘙痒病的易感性.方法 参照GenBank上已经发表的绵羊PRNP序列设计引物,对岩羊的PRNP序列进行扩增,基因测序后与GenBank上的11种哺乳动物的PRNP序列比对和分析.结果 测序结果显示岩羊PRNP由771个核苷酸构成,编码256个氨基酸.序列分析结果显...     

Competition between strains of scrapie depends on the blocking agent being infectious

Compton White mice (Sincs7) were injected twice intraperitoneally, first with the 22A strain of scrapie agent (in brain homogenates) and then, after 105 days, with the 22C strain. Incubation periods were calculated from the time of the first injection. The experiment was designed so that, with no interaction between strains, the second strain (22C) should have produced cases about 300-350 days after the first injection, depending on the dose of 22C. This was well before the limit of 470 days set by the mean incubation period minus 3 SD of 22A alone: a limit which was used to distinguish 22C from 22A clinical cases. In fact, 22A blocked 22C as shown by (i) the lengthening of 22C incubation periods, (ii) the reduced proportion of cases due to 22C, and (iii) the reduced effective titer of 22C. The blocking efficiency of 22A was not greatly reduced by physicochemical treatments that had little or no effect on its infectivity by the intraperitoneal route. However, treatment of 22A homogenates with 6 M urea virtually eliminated infectivity and also abolished blocking ability. It is concluded that competition depends on the infectivity of the scrapie strain used for blocking.     

In vitro interaction of scrapie agent and mouse peritoneal macrophages

Scrapie brain homogenate was mixed with mouse peritoneal macrophages in vitro. After 2 h of incubation at 37 degrees, a portion of the scrapie infectivity was associated with macrophages. In contrast, very little infectivity was associated with kidney cells that had been exposed to scrapie brain homogenate. After incubation of the scrapie brain homogenate-macrophage mixture at 4 degrees rather than 37 degrees, a reduced quantity of infectivity was associated with the macrophages. These result show that in vitro incubation, the scrapie agent was associated with macrophages, and the data suggest that phagocytic activity was involved.     

Partitioning of human and sheep forms of the pathogenic prion protein during the purification of therapeutic proteins from human plasma

BACKGROUND : Therapeutic proteins derived from human plasma and other biologic sources have demonstrated an excellent safety record relative to the potential threat of transmissible spongiform encephalopathy (TSE) transmission. Previously, hamster‐adapted scrapie was used as a model agent to assess TSE clearance in purification steps leading to the isolation of biopharmaceutical proteins. The current study investigated the validity of hamster scrapie as a model for human TSE clearance studies. The partitioning of the pathogenic forms of the prion protein associated with human variant CJD (PrP^vCJD), human sporadic CJD (PrP^sCJD) and Gerstmann‐Sträussler‐Scheinker (PrP^GSS) syndrome was compared to the partitioning of hamster scrapie (PrP^Sc) in three plasma protein purification steps. Sheep scrapie (PrP^Sc) was similarly evaluated. STUDY DESIGN AND METHODS : The starting materials for three plasma protein purification steps, cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation, were spiked with brain homogenates containing human PrP^vCJD, human PrP^sCJD, human PrP^GSS, sheep PrP^Sc, and hamster 263K PrP^Sc. The partitioning of the pathogenic form of the PrP was analyzed. RESULTS : Clearance of the pathogenic form of the PrP was measured relative to the effluent fraction. Regardless of the source of the pathogenic prion, clearance was similar to hamster PrP^Sc. A nominal amount of clearance (approx., 1 log), an intermediate amount of clearance (approx., 2 log), and a substantial amount of clearance (≥3 log) were observed for the cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation steps, respectively. In the latter step, no PrP was detected in the effluents. CONCLUSIONS : These data demonstrate that human prions, including vCJD prions, can be removed during the purification of human therapeutic proteins and indicate that partitioning of human prions is similar to that observed in the hamster scrapie model.     

A porphyrin increases survival time of mice after intracerebral prion infection

Prion diseases, including scrapie, are incurable neurodegenerative disorders. Some compounds can delay disease after a peripheral scrapie inoculation, but few are effective against advanced disease. Here, we tested multiple related porphyrins, but only Fe(III)meso-tetra(4-sulfonatophenyl)porphine injected into mouse brains after intracerebral scrapie inoculation substantially increased survival times.     

Suppression of scrapie infection in mice by heteropolyanion 23, dextran sulfate, and some other polyanions.

Studies of polyanions that suppress scrapie have been done to pinpoint the cell types in the lymphoreticular system which are important in pathogenesis and to suggest possible prophylactic or therapeutic strategies for the unconventional slow viruses. A regime of three daily injections of the inorganic heteropolyanion HPA-23 reduced the effective scrapie dose by more than 99%; i.e., some mice survived peripherally injected doses of 100 50% lethal dose units. The effect was greatest when the first dose of HPA-23 was given 4 h after injecting scrapie, but it declined rapidly as this interval was increased, and there was virtually no effect 2 days after infection. A single dose of high-molecular-weight organic polyanions such as carrageenan or dextran sulfate (DS-500) greatly reduced (i.e., greater than 99%) the efficiency of scrapie infection. In contrast to HPA-23, DS-500 was equally effective whether given 4 days before or 8 h after the time of infection. The antiscrapie effect of DS-500 appeared to be independent of its activity as a B-cell mitogen and of its ability to produce a cytotoxic blockade of phagocytic cells. DS-500 probably caused the aggregation and loss from blood of scrapie inoculum which was present immediately after injection, but it had additional effects on scrapie at later times.     

MS-8209, a new amphotericin B derivative, provides enhanced efficacy in delaying hamster scrapie.

To test the efficacy of a new amphotericin B derivative, MS-8209, in delaying scrapie, hamsters were infected intracerebrally with the 263K scrapie agent and treated with MS-8209 either early in the course of the disease or continuously. The results show that (i) all treatments lengthened the incubation period of hamster scrapie, (ii) continuous treatment with MS-8209 doubled the length of the incubation period compared with that observed in infected, untreated animals, and (iii) all treatments delayed the accumulation of a proteinase-resistant prion protein and glial fibrillary acidic protein in the brain. These findings suggest that MS-8209 is a powerful tool for investigating the pathogenesis of transmissible subacute spongiform encephalopathies.     

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

Recently, pathological prion protein PrP(Sc), the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrP(Sc) accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrP(Sc) in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrP(Sc) was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrP(Sc) and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from "Prions in skeletal muscle" and provide new insights into the pathophysiological spread of TSE agents through the body.     

Evidence of ssDNA in tubulofilamentous particles: their relationship to scrapie-associated fibrils.

Abnormal tubulofilamentous particles were identified by electron microscopy using a simple touch negative staining technique from brains of mice infected with four strains of the scrapie agent. Treatment by three proteolytic enzymes and subsequent treatment with DNase and mung bean nuclease of grids prepared from the infected animals confirmed previous observations that the tubulofilamentous particles observed in scrapie-effected brains are complex structures. The core of the tubulofilamentous particle scrapie-associated fibrils was revealed by treatment with SDS. Treatment with proteolytic enzymes and subsequent treatment with DNase or mung bean nuclease or S1 nuclease also revealed typical and transitional stages of scrapie-associated fibrils. However, treatment with RNase A had no effect. The data suggest that nucleic acid is a single-stranded DNA protected by a protein coat.     

动力相关蛋白Drp1在羊瘙痒因子139A感染小鼠脑组织中变化的研究

目的 研究动力相关蛋白1(Drp1)在羊瘙痒因子139A感染的小鼠脑组织中的变化。方法 采用Western Blot方法检测Drp1在羊瘙痒因子139A感染的脑组织匀浆中的含量变化及其亚硝基化水平。采用组织免疫荧光方法检测Drp1在脑组织中的分布。结果 在羊瘙痒因子139A感染终末期小鼠脑组织匀浆中,Drp1总量略有降低。对感染不同时间点的动态分析结果显示Drp1含量呈逐渐降低趋势,终末期稍有回升。感染终末期脑组织中亚硝基化水平明显增高。组织免疫荧光显示正常和羊瘙痒因子139A感染终末期小鼠脑组织中,Drp1与神经元细胞存在共定位现象。结论 Drp1在小鼠中枢神经系统中主要分布于神经元细胞,在羊瘙痒因子139A感染的小鼠脑组织中,Drp1总量略有降低,另外亚硝基化水平明显增高,提示在羊瘙痒病感染的小鼠脑组织中,线粒体动力相关蛋白的表达量及翻译后修饰水平出现了变化,在感染过程中起着重要作用。