Survival of scrapie virus after 3 years' interment.

Supernatant fluid from a scrapie-infected hamster brain homogenate was mixed with soil, packed into perforated petri dishes that were then embedded within soil-containing pots, and buried in a garden for 3 years. Between 2 and 3 log units of the input infectivity of nearly 5 log units survived this exposure, with little leaching of virus into deeper soil layers. These results have implications for environmental contamination by scrapie and by similar agents, including those of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease.

Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer.

A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.

A simple and effective method for inactivating virus infectivity in formalin-fixed tissue samples from patients with Creutzfeldt-Jakob disease.

We fixed brains from hamsters infected with scrapie virus in (1) formalin, (2) phenol-saturated formalin, (3) formalin with a 1-hour immersion in formic acid, or (4) phenol-saturated formalin with a 1-hour immersion in formic acid. In addition, we used the formalin-formic acid procedure on brains from mice infected with the virus of Creutzfeldt-Jakob disease. Formic acid proved superior to phenol in respect to both disinfection and tissue preservation, almost completely eliminating virus infectivity in sections that were histologically indistinguishable from formalin-fixed material. The inclusion of a formic acid step in routine formaldehyde tissue fixation will thus provide histologic sections of excellent quality, and virtually eliminate the risk of handling infectious material in the subsequent neuropathologic processing of tissues from patients with CJD.

Organ-specific modification of the dose-response relationship of scrapie infectivity.

The dose-response relationships of scrapie strain 263K-infected hamster brain and spleen homogenates were compared to determine if intracerebral end-point titrations of infectivity in these homogenates were measures of the same pathogenic phenomenon. Analysis of the dose-response curves indicated that the average increase in incubation period per 10-fold dilution (i.e., the dilution kinetics) of brain infectivity was significantly different from that of spleen infectivity. This difference contradicted the assumption that the same pathogen or pathogenic mechanisms were responsible for producing disease in each titration. Therefore, the end-point titrations and infectivity titers of the brain and spleen homogenates were measures of two different phenomena. Subsequent passage of a scrapie-infected spleen homogenate demonstrated that the dose-response relationship of scrapie infectivity in this agent-host system was dependent on the organ titrated, not the tissue source or inoculation route of previous passage.

Amphotericin B: a novel class of antiscrapie drugs.

Amphotericin B (AmB) has been able to lengthen the incubation period of intracerebrally (ic) scrapie-injected hamsters to 45 d. This article reports a linear relationship between AmB doses and the duration of the incubation periods of ic-treated animals compared with controls, a greater effect of AmB treatment administered 2 w before or the same day of ic scrapie incubation, and the ineffectiveness of mepartricin, an AmB analogue, in prolonging the incubation period of ic scrapie-injected hamsters. The beneficial effect of AmB appears due to a delay in the replication of the scrapie agent in the brain of infected hamsters. Moreover, AmB suppresses scrapie replication in the spleen of treated animals. Three hypotheses may explain these results: (1) AmB alters a hypothetical scrapie receptor, preventing the entry of the agent into central nervous system (CNS) target cells; (2) AmB interferes with mechanisms involved in scrapie replication; (3) AmB prevents the formation and accumulation of a scrapie-specific amyloid protein responsible for the disease. Whatever the mechanism of action, AmB is the only currently available drug to modify experimental CNS scrapie infection, so AmB is proposed as a novel class of antiscrapie drugs.

Levels of infectivity in the blood throughout the incubation period of hamsters peripherally injected with scrapie.

Viremia is found in intraperitoneally scrapie-injected hamsters. The absence of a viremic peak before the beginning of scrapie replication in the brain suggests either that the spread of the agent to the brain is not via the blood or that early after infection, circulating monocytes carry the agent to the brain where it remains silent until the neural cells start replicating it.

Adrenal involvement in scrapie-induced obesity.

In previous studies we found an increase in body weight during the preclinical phase of disease in certain scrapie strain-mouse strain combinations. The effect was augmented by injection into the hypothalamus. In the present study, we found an increase in food consumption (compared to the normal mouse brain injection group) for both the 139A and ME7 scrapie groups, although only the ME7 group showed an increase in body weight. In a scrapie strain-mouse strain combination that showed an increase in body weight, the adrenal gland was the only organ that showed a significant increase in weight. The titer of scrapie in the adrenals was comparatively low. Adrenalectomy prevented the increase in body weight in two strains of mice injected with the ME7 scrapie strain. The results suggest that scrapie-induced obesity depends on an effect of scrapie on the hypothalamic-pituitary-adrenal axis.

Pathogenesis of scrapie: study of the temporal development of clinical symptoms, of infectivity titres and scrapie-associated fibrils in brains of hamsters infected intraperitoneally.

After an intraperitoneal infection of hamsters with scrapie agent, early low and constant titres of about 100 LD50/brain between days 10 to 50 were followed by a dramatic increase to maximum levels of 3 X 10(9) LD50/brain within about 15 days. The plateau of maximum infectivity remained unchanged from day 70 to the time of the first and final signs of disease at 95 and 123 days post-infection, respectively. Scrapie-associated fibrils (SAF) as measured by immunoblotting of SAF protein could not be detected before 79 days post-infection even when a total brain was used for analysis. Subsequently, the concentration of SAF increased gradually by about 100,000-fold until the time of clinical disease. The kinetics suggest a virus-induced amyloidosis of the brain as the cause of disease.

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.

Agent replication dynamics in a long incubation period model of mouse scrapie.

The dynamics of scrapie agent replication in brain and spleen following intracerebral or intraperitoneal infection were investigated in a model with particularly long incubation periods [IM mice (Sincp7) infected with 87V scrapie]. In contrast to other mouse scrapie models previously investigated, there was no delay in onset of replication ('zero phase') in spleen using either route of infection, For both routes infectivity levels reached a plateau in spleen, which was maintained for at least 250 days in intraperitoneally infected mice. An infectivity plateau was also apparent in brain following intracerebral infection, suggesting that there may be constraints on agent replication in brain during the later part of the incubation period. In addition, the efficiency of the intraperitoneal route to produce disease was found to be particularly low for this model.

Transmission of scrapie in hamsters.

Hamsters developed scrapie 100-160 days after eating either scrapie-infected hamsters or infected brain. The clinical signs and neuropathology of scrapie transmitted by cannibalism were identical to those observed after intracerebral or intraperitoneal inoculation of the agent. Oral transmission of scrapie appears to be extremely inefficient. Cannibalism requires a dose of the scrapie agent of approximately 10(9) times greater than that needed to produce the disease by intracerebral injection for comparable periods of incubation. These results provide compelling evidence for oral transmission of scrapie and may offer new insights into the spread of kuru by cannibalism among the Fore people and their neighbors. The extreme inefficiency of oral infection with scrapie might also have implications for understanding the sporadic occurrence and worldwide distribution of Creutzfeldt-Jakob disease.

In vitro studies with the scrapie agent.

Persistent infection with the scrapie agent has been established in L cells. The agent was propagated in homogenates of the cell line designated L-S. The L-S cells showed slower growth rate and morphological changes like pycnosis of nuclei and vacuolization of their cytoplasm. Cytogenetic analysis revealed rearrangement of chromosomes in L-S cells. In the course of passaging the number of cells with the characteristic marker chromosome decreased. Along with this cells were found with deletion of one arm of the marker chromosome. In addition, 3 new marker chromosomes were detected in infected cells, suggesting the influence of the scrapie agent on cytogenetic processes in scrapie-carrier cultures. The infectious activity of nucleic acids isolated from L-S cells was determined in BALB/c mice inoculated with untreated, DNase-treated and pronase-treated nucleic acid preparations. A slightly decreased infectious activity has been noted after DNase and pronase treatments.

Experimental infection of fetal and newborn Suffolk sheep with scrapie virus.

Fetal (n = 21) and newborn (n = 7) Suffolk sheep were inoculated with scrapie virus isolated from other Suffolk sheep. Twenty fetuses, 76 to 109 days of gestational age, were inoculated IM in the neck through the uterine wall and were examined for virus 47 to 322 days later by mouse inoculation. Scrapie virus was not detected before 254 days of age; only traces of virus were detected in 3 of 7 lambs examined thereafter (2 at 254 days of age and 1 at 322 days of age). Virus was limited to the supra-pharyngeal, prescapular, and mesenteric lymph nodes. Seven lambs were inoculated into the palatine tonsils with scrapie virus as newborns (3 to 12 days old) and were examined for virus when they were 147 to 210 days old. Virus was not detected in the lymphoreticular tissues or terminal portion of ileum of any lamb. Failure to find scrapie virus in these lambs and in most lambs inoculated as fetuses might indicate few had became infected. However, if most lambs and fetuses had become infected, the long zero phase of the infection could have accounted for failure to find scrapie virus in many of them examined too soon after inoculation. The limited findings of this study indicate that efforts to demonstrate prenatal or neonatal transmission of scrapie by detecting virus are hampered by the slowness of its replication.

In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells.

A rat phaeochromocytoma cell line, termed PC12, was used to study scrapie replication. These cells, in response to the addition of nerve growth factor (NGF), exhibit a number of neuronal properties including morphological differentiation, electrophysiological responsiveness, and neurotransmitter synthesis. Cultures were exposed to scrapie brain homogenate (strain 139A), harvested every week for up to 6 weeks, and assayed for scrapie infectivity. Scrapie replication in vitro was monitored by injecting scrapie agent-exposed NGF-treated PC12 cells into mice and measuring time intervals from injection to onset of clinical symptoms. Mouse incubation periods vary inversely with the amount of scrapie infectivity present. Cells harvested at 7 and 14 days after exposure to scrapie agent showed a decrease in the level of infectivity followed by an increase at subsequent time points. The increase in scrapie infectivity from early to late time intervals after agent exposure clearly indicated replication in vitro. A fusion agent was not necessary to establish infection, and the addition of mouse peritoneal macrophages caused a reduction in the yield of infectivity per culture. Examination of cells by phase-contrast microscopy failed to reveal any cytopathology.

Virus like sensitivity of the scrapie agent to heat inactivation.

The resistance of the infectious agent of scrapie disease to sterilization at 100 degrees or 121 degrees C is reputed to be inconsistent with the structure of conventional viruses. However, in kinetic studies the majority of hamster scrapie strain 263K infectivity was (like that of previously characterized viruses) rapidly inactivated at temperatures of 100 degrees C or greater. Small resistant subpopulations remained. Similar heat-resistant subpopulations were observed at 60 degrees C for phage lambda but only in the presence of brain homogenate. Brain homogenate may also confer stability to small subfractions of scrapie infectivity. Such refractory subpopulations cannot be used to make structural inferences that are properly obtained from the behavior of the majority population as revealed in the initial inactivation.

Genetic control of scrapie and Creutzfeldt-Jakob disease in mice.

Genetic control of experimental scrapie and Creutzfeldt-Jakob disease (CJD) was studied in inbred strains of mice by measuring the times from intracerebral inoculation with the agents to the onset of neurological dysfunction. Every strain of mice examined was susceptible to infection; however, a wide range of incubation times was found for both scrapie and CJD. New Zealand (NZ) mice, which eventually develop an autoimmune disorder, were inoculated intracerebrally with 10(6) ID50 units of the scrapie agent in a Chandler isolate. NZW mice showed incubation periods of less than 95 days; this is the shortest period recorded for any murine host with scrapie. In NZB and NZB X W F1 mice, the incubation periods were approximately 130 days and were similar to those in BALB/c and C57BL mice. Male and female NZ mice exhibited scrapie incubation periods of the same length. Similar results were obtained when B10.Q and C57BL/6J mice were inoculated intracerebrally with 10(4) ID50 units of the CJD agent in a K.Fu. isolate. These observations define a genetic locus or loci controlling the length of scrapie and CJD incubation periods; alleles coding for longer incubation times appear to be autosomal dominant. When congenic mice with a C57BL/10J background differing only in their H-2 haplotypes were studied, the results showed that the D subregion of the H-2 complex played a central role in controlling the length of the CJD incubation period. The q allele at the D subregion resulted in shorter incubation times, whereas the d allele resulted in long incubation times. The p, s, b, and k alleles gave intermediate incubation times. We propose the symbol PID-1 for designating this genetic locus which is located within the D subregion of the major histocompatibility (H-2) complex on murine chromosome 17. In addition, observations on congenic mice provide evidence for the influence of sex on CJD incubation periods. In some strains of inbred mice, males showed significantly shorter incubation periods compared with those for females with experimental CJD. These studies with inbred mice have defined previously unrecognized genes that control the length of scrapie and CJD incubation periods.

Study of early stages of the pathogenesis of scrapie in experimentally infected mice.

Virological, histological, and electron microscopic methods were used to study the early manifestations of infection in brains and spleens of mice experimentally infected with the scrapie agent. A statistically significant increase in the spleen weight was demonstrated during the incubation period; this occurred only after intracerebral (i.c.) inoculation of the scrapie-containing brain suspensions and subcutaneous (s.c.) inoculation of the scrapie-containing spleen suspensions. No differences were observed between the scrapie agents replicating in the brain or spleen. In the brain at early stages of the incubation period, ultrastructural changes were observed in pre- and post-synaptic areas. The importance of these findings for understanding of the pathogenesis of subacute transmissible spongiform encephalopathy (STSE) is discussed.

Identification of a protein that purifies with the scrapie prion.

Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

Novel proteinaceous infectious particles cause scrapie.

After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.

Effect of mouse peritoneal macrophages on scrapie infectivity during extended in vitro incubation.

Mouse peritoneal macrophages were exposed to scrapie (ME7 strain) brain homogenate in vitro for 2 h at 37 degrees. The samples were assayed for infectivity by analysis of scrapie incubation periods and their values compared to those obtained after extended (1-28 days) in vitro incubation. The scrapie incubation periods for scrapie agent-macrophage mixtures which had undergone extended in vitro incubation were longer than for mixtures assayed after a 2-hour exposure to the scrapie agent. The difference in scrapie incubation periods was more dramatic when residual scrapie was eliminated by washing the cells after the 2-hour exposure to brain homogenate. The scrapie incubation period also increased following in vitro incubation of ME7 in the absence of cells; however, the changes were less than those observed for scrapie agent-macrophage mixtures. In a culture in which cells had been destroyed by UV irradiation after exposure to the scrapie agent, there was more scrapie infectivity than in a comparable culture of untreated, scrapie-exposed macrophages. These results show that scrapie infectivity decreases with extended incubation of scrapie-exposed macrophages, and the data suggest that macrophages can inactivate the scrapie agent in vitro.