Pathology of SSLOW, a transmissible and fatal synthetic prion protein disorder, and comparison with naturally occurring classical transmissible spongiform encephalopathies.

AIMS: Naturally occurring transmissible spongiform encephalopathies (TSEs) accumulate disease-specific forms of prion protein on cell membranes in association with pathognomonic lesions. We wished to determine whether synthetic prion protein disorders recapitulated these and other subcellular TSE-specific changes. METHODS: SSLOW is a TSE initiated with refolded synthetic prion protein. Five terminally sick hamsters previously intracerebrally inoculated with third passage SSLOW were examined using light and immunogold electron microscopy. RESULTS: SSLOW-affected hamsters showed widespread abnormal prion protein (PrP(SSLOW) ) and amyloid plaques. PrP(SSLOW) accumulated on plasma lemmas of neurites and glia without pathological changes. PrP(SSLOW) also colocalized with increased coated vesicles and pits, coated spiral membrane invaginations and membrane microfolding. PrP(SSLOW) was additionally observed in lysosomes of microglial cells but not of neurones or astrocytes. CONCLUSIONS: PrP(SSLOW) is propagated by cell membrane conversion of normal PrP and lethal disease may be linked to the progressive growth of amyloid plaques. Cell membrane changes present in SSLOW are indistinguishable from those of naturally occurring TSEs. However, some lesions found in SSLOW are absent in natural animal TSEs and vice versa. SSLOW may not entirely recapitulate neuropathological features previously described for natural disease. End-stage neuropathology in SSLOW, particularly the nature and distribution of amyloid plaques may be significantly influenced by the early redistribution of seeds within the inoculum and its recirculation following interstitial, perivascular and other drainage pathways. The way in which seeds are distributed and aggregate into plaques in SSLOW has significant overlap with murine APP overexpressing mice challenged with Aß.

Characterization of a US sheep scrapie isolate with short incubation time.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US-origin sheep scrapie, that results in incubations nearly threefold shorter. The present study documents clinicopathologic findings and the distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanatized within an average of 7.7 months postinoculation (MPI). Sheep that had valine/valine or alamine/valine at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanatized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

Characterization of scrapie-infected and normal hamster blood as an experimental model for TSE-infected human blood.

Ideally, the distribution and separation properties of blood-associated infectivity in humans infected with transmissible spongiform encephalopathies (TSEs) would be investigated using endogenously infected human blood. However, technical limitations prevent these studies. We report here an extensive comparison of component separations of TSE-infected and normal hamster blood with normal human blood to evaluate its suitability as a model for TSE-infected human blood. Two leukotrap filtration systems were used to assess the similarities and differences in cell separation and distribution between human and hamster blood. Human-sized units of scrapie-infected hamster whole blood pools were produced, leukoreduced and processed following current standard blood bank procedures. Three centrifugation steps were performed, and all but one centrifugation resulted in cell separations both similar to those obtained with human blood and in compliance with the American Association of Blood Banks standards. Analysis of white blood cell removal by leukoreduction indicated that all filters performed within specification. The only deviation from human blood was for hamster platelets, which were not retained by the whole blood leukoreduction filter and partitioned together with red blood cells when blood was centrifuged to produce platelet rich plasma. This study indicates that hamster blood is a realistic substitute for human blood for investigations of the component distribution of TSE infectivity in blood.

Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.

Inactivation of viral and prion pathogens by gamma-irradiation under conditions that maintain the integrity of human albumin.

BACKGROUND AND OBJECTIVES: The administration of therapeutic plasma protein concentrates has been associated with the real risk of transmitting viral diseases and the theoretical risks of prion transmission. Our objective was to determine if gamma-irradiation can inactivate viral or prion infectivity without damaging a protein biotherapeutically. MATERIALS AND METHODS: Human albumin 25% solution, spiked with four model viruses (including porcine parvovirus) or with brain homogenate from scrapie-infected hamsters, was gamma-irradiated at constant low-dose rates and assayed for viral and prion infectivity or for albumin integrity. RESULTS: At a radiation dose of 50 kGy, viruses were inactivated by >/= 3.2 to >/= 6.4 log10 and scrapie by an estimated 1.5 log10, whereas albumin was only moderately aggregated and fragmented. CONCLUSIONS: gamma-Irradiation can preferentially inactivate viral and prion pathogens without excessive damage to albumin structure.

The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy.

BACKGROUND: The administration of blood components from donors who subsequently develop Creutzfeldt-Jakob disease has raised the issue of blood as a possible vehicle for iatrogenic disease. STUDY DESIGN AND METHODS: We examined infectivity in blood components and Cohn plasma fractions in normal human blood that had been "spiked" with trypsinized cells from a scrapie-infected hamster brain, and in blood of clinically ill mice that had been inoculated with a mouse-adapted strain of human transmissible spongiform encephalopathy. Infectivity was assayed by intracerebral inoculation of the blood specimens into healthy animals. RESULTS: Most of the infectivity in spiked human blood was associated with cellular blood components; the smaller amount present in plasma, when fractionated, was found mainly in cryoprecipitate (the source of factor VIII) and fraction I+II+III (the source of fibrinogen and immunoglobulin); almost none was recovered in fraction IV (the source of vitamin-K-dependent proteins) and fraction V (the source of albumin). Mice infected with the human strain of spongiform encephalopathy had very low levels of endogenous infectivity in buffy coat, plasma, cryoprecipitate, and fraction I+II+III, and no detectable infectivity in fractions IV or V. CONCLUSION: Convergent results from exogenous spiking and endogenous infectivity experiments, in which decreasing levels of infectivity occurred in cellular blood components, plasma, and plasma fractions, suggest a potential but minimal risk of acquiring Creutzfeldt-Jakob disease from the administration of human plasma protein concentrates.

Analysis of risk to biomedical products developed from animal sources (with special emphasis on the spongiform encephalopathy agents, scrapie and BSE).

Factors that must be considered in estimations of risk from exposure to adventitious contaminants of animal derived biologicals include: (i) the use of the product; (ii) the routes of administration and exposure to potential pathogens; (iii) the source of animal(s) and their history and maintenance; (iv) the tissue(s) used in the product and their likelihood of harbouring or being contaminated by an agent; (v) the methods by which the animal is slaughtered and the tissue(s) collected; (vi) the production process including steps that remove an agent either by inactivation or physical separation; (vii) the disposal of an agent that is sequestered but not inactivated; (viii) flow control, barriers, and between-batch cleaning; (ix) batch size; and (x) validations and quality assurance monitoring. The transmissible spongiform encephalopathy (TSE) agents, scrapie and bovine spongiform encephalopathy (BSE) are among the most difficult to detect and remove from animal tissues. As such they constitute an especially stringent challenge for viral clearance.

Clostridium difficile infection in adult hamsters.

Diarrhea was encountered in a group of adult female golden Syrian hamsters (Mesocricetus auratus) used for titrating the scrapie agent. Ninety percent of the cases occurred in animals over 210 days old even though animals of all age groups lived in the colony concurrently. The cause of diarrhea was investigated in both uninoculated animals and those receiving greater than a limiting dilution of scrapie infectivity, i.e., animals that were not expected to contract the experimental scrapie disease. Three forms of diarrhea were observed. The most commonly encountered was profuse and watery. A chronic form presented with semiformed, thin fecal material smearing the retroperitoneal region. Hemorrhagic diarrhea was observed rarely. Mortality was high among animals with acute watery or hemorrhagic diarrhea. Animals with semiformed soft stools were dehydrated, had a roughened hair-coat, and hunched back. Cardinal lesions were necrosis, inflammation, and mucosal hyperplasia of the cecum and colon and cholangiohepatitis with amyloid deposition. Diffuse renal amyloidosis was present in chronic cases. Toxigenic, cytotoxin B-positive Clostridium difficile was isolated from a majority of affected animals. Cytotoxin B was also present in cecal homogenates of diarrheic animals with C. difficile. The pathological and microbiologic findings indicated a typhlitis and colitis in adult hamsters that was associated with C. difficile infection.

Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

Transmission in NFS/N mice of the heritable spongiform encephalopathy associated with the gray tremor mutation.

It has been shown that the autosomal recessive mutation, gray tremor (gt) was associated in the homozygous state (gt/gt) with a rapidly fatal spongiform encephalopathy. Heterozygotes (+/gt) developed mild asymptomatic spongiform brain lesions as did recipient inbred mice inoculated with gt/gt brain homogenates, some of whom also showed behavioral abnormalities [Sidman, R. L., Kinney, H. C. & Sweet, H. O. (1985) Proc. Natl. Acad. Sci. USA 82, 253-257]. In these studies, inbred NFS/N mice inoculated intracerebrally at birth or as adults with gt/gt or first passage gt brain homogenates developed a progressive disease characterized by tremor, ataxia, and spasticity. The symptoms were milder and more slowly progressive than in the gt/gt homozygote, in the paralytic syndrome that followed neonatal inoculation of NFS/N mice with a wild murine leukemia virus (Cas-Br-M MuLV), or in the rapidly progressive ataxia and terminal bradykinesia that followed scrapie inoculation of NFS/N mice. The noninflammatory spongiform encephalopathy in affected NFS/N mice resembled that observed in gt/gt homozygotes, +/gt heterozygotes, and asymptomatic recipient inbred mice inoculated with gt/gt brain homogenates. Neither infectious MuLV nor MuLV proteins were detected in gt/gt brain homogenates or in affected recipient mouse brains. Scrapie-associated fibrils, readily identifiable in subcellular fractions of brains from scrapie-inoculated NFS/N mice, were not detected in similar brain fractions from NFS/N mice inoculated with gt brain homogenates. These results confirm and extend the suggestion that gt spongiform encephalopathy has both heritable and transmissible properties. Moreover, the transmissible agent of gt disease differs from both Cas-Br-M MuLV and scrapie in its disease-inducing properties in NFS/N mice. The capacity of NFS/N mice to express transmitted gt encephalopathy as clinical disease, to rapidly express Cas-Br-M MuLV spongiform encephalomyelopathy, and to develop mouse-adapted scrapie after a very short incubation time suggest a distinct sensitivity of NFS/N mice to transmissible spongiform encephalopathy.

Infection-specific particle from the unconventional slow virus diseases.

Scrapie-associated fibrils, first observed in brains of scrapie-infected mice, were also observed in scrapie-infected hamsters and monkeys, in humans with Creutzfeldt-Jakob disease, and in kuru-infected monkeys. These fibrils were not found in a comprehensive series of control brains from humans and animals affected with central nervous system disorders resulting in histopathologies, ultrastructural features, or disease symptoms similar to those of scrapie, kuru, and Creutzfeldt-Jakob disease. These fibrils are also found in preclinical scrapie and in the spleens of scrapie-infected mice; they are a specific marker for the "unconventional" slow virus diseases, and may be the etiological agent.

The sequential development of spongiform change and gliosis of scrapie in the golden Syrian hamster.

The lesion profiles of spongiform change and gliosis in the hamster occurring after intracerebral (IC) inoculation of scrapie virus, are calculated and compared to the lesion profile of spongiform change of scrapie in mice and of scrapie and Creutzfeldt-Jakob disease (CJD) in the squirrel monkey. The profile of scrapie in hamsters differs considerably from that of a closely related strain of scrapie in mice, and both differ from scrapie and CJD in the squirrel monkey. These differences emphasize the effect of the host on the distribution of pathological changes in these unconventional virus infections. The sequential development of the lesions in the hamster shows that the earliest changes are detectable before the onset of clinical disease 49-57 days after inoculation, as assessed by light microscopy. Gliosis is detectable by indirect immunofluorescence 35-39 days after inoculation by use of a monoclonal antibody directed against astrocytes.

Scrapie infectious agent is virus-like in size and susceptibility to inactivation.

The virions of all known viruses are composed of small amounts of genomic nucleic acid enveloped by proteins and other macromolecules. The aetiological agents of scrapie disease and the other subacute spongiform virus encephalopathies (SSVE), a group of slow, fatal degenerative diseases of the central nervous system, are, based on their resistance to sterilization and on indirect measurements suggesting subviral size, thought to have non-viral structures (see refs 1-3 for reviews). The kinetic studies reported here demonstrate that scrapie's resistance to many inactivants is limited to small subpopulations of the total infectivity, the majority population being highly sensitive to inactivation. Moreover, control inactivations of conventional viruses provide examples of both scrapie-like resistant subpopulations and complete insensitivity to virucidal agents, especially when those viruses, like scrapie, are suspended in hamster brain homogenate. Virus controls further establish that the ability of the scrapie agent to penetrate dilute agarose-acrylamide electrophoretic gels is shared by conventional viruses. Direct comparison of scrapie's resistance to ionizing radiation with the resistances of other viruses places scrapie with the smaller viruses, as opposed to requiring a subviral size as claimed.

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.