Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent.

To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellum, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scrapie, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Scrapie-associated PrP accumulation and its inhibition: revisiting the amyloid-glycosaminoglycan connection.

An abnormal protease-resistant isoform of the protein PrP accumulates in the brain of hosts with transmissible spongiform encephalopathies (TSEs) and appears to be centrally involved in TSE pathogenesis. Studies with scrapie-infected tissue culture cells have indicated that this abnormal PrP is formed from an apparently normal precursor on the plasma membrane or along an endocytic pathway to the lysosomes. Inhibitors of protease-resistant PrP accumulation might serve as tools for studying the basic mechanism of protease-resistant PrP formation and as potential drugs for TSE therapy. Using scrapie-infected neuroblastoma cells to screen for such compounds in vitro, we found that the amyloid binding dye Congo red and certain sulfated glycans potently inhibited the accumulation of protease-resistant PrP in scrapie-infected cells without apparent effects on the metabolism of the normal isoform. The relative potencies of the sulfated glycans corresponded with their previously determined anti-scrapie activities in vivo, suggesting that the prophylactic effects of sulfated polyanions may be due to inhibition of protease-resistant PrP accumulation. Since protease-resistant PrP amyloid is known to contain sulfated glycosaminoglycans, as do other naturally derived amyloids, we hypothesize that these sulfated inhibitors competitively block binding between PrP and endogenous glycosaminoglycans that is important for its accumulation in a protease-resistant, potentially amyloidogenic state. Drugs which interfere with this (pre)amyloid-glycosaminoglycan interaction may be useful for treating a variety of amyloidoses.

Comparative analysis of scrapie agent inactivation methods.

A scrapie-infected hamster brain homogenate was subjected to several different potential inactivation methods. Methods included autoclaving for various lengths of time, either alone or in combination with different concentrations of sodium hydroxide or LpH, an aqueous acid phenolic derivative (Calgon Vestal Laboratories in St. Louis, MO). Inactivation treatments utilizing either NaOH or LpH alone were also evaluated. It was determined that several of the treatments inactivated all of the detectable infectivity.

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update.

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease-sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.

Immunohistochemical detection of prion protein in sheep with scrapie.

Prion protein (PrP), which is involved in the pathogenesis of scrapie, occurs in 2 forms. The form extracted from scrapie brain is protease resistant (PrP-res), whereas PrP from normal brain is protease sensitive (PrP-sen). This study examined whether PrP-res could be detected in brains of sheep with scrapie by immunohistochemistry (IHC). A suitable IHC procedure was developed using brain tissue from hamsters that had been inoculated with the transmissible mink encephalopathy agent. Tissue samples were fixed in PLP (periodate, lysine, paraformaldehyde) that contained paraformaldehyde at a concentration of 0.125%. Before application of the IHC technique, tissue sections were deparaffinized and treated with formic acid to simultaneously enhance PrP-res immunoreactivity and degrade PrP-sen. Primary antibody was obtained from a rabbit immunized to PrP-res extracted from brains of mice with experimentally induced scrapie. Brain from 21 sheep with histopathologically confirmed scrapie were examined by IHC. In all 21 brains, PrP-res was widely distributed throughout the brain stem. Staining was particularly intense in neuronal cell bodies and around blood vessels. The IHC technique successfully detected PrP-res in brain samples that had been frozen or that were severely autolyzed before fixation in PLP. Brains from 11 scrapie-suspect sheep that were not considered histologically positive were also examined by IHC. PrP-res was found in 4 of these brains. Sections of brains from 14 clinically normal sheep did not have detectable PrP-res. Results of this study indicate that IHC detection of PrP-res is equivalent, and perhaps superior, to histopathology for the diagnosis of scrapie in sheep. Furthermore, IHC is applicable to tissues that have autolytic changes or processing artifacts that prevent satisfactory histopathologic evaluation for lesions of scrapie.

Experimental infection of sheep and goats with transmissible mink encephalopathy virus.

In a study to learn more about the pathogenicity of transmissible mink encephalopathy virus for the natural hosts of scrapie, 20 Cheviot sheep and 19 dairy goats were inoculated intracerebrally with the Idaho strain of the virus. Five sheep and nine goats became affected with a progressive neurological disease. The incubation period in the sheep varied from 45 to 80 months (mean, 65 months) and in the goats from 31 to 40 months (mean, 35 months). Except for degeneration of the cerebral cortex (neocortex), the disease was indistinguishable clinically and neurohistologically from scrapie. During two more passages of the virus in goats, the incubation period was shortened to 12 to 15 months, the morbidity rate rose to 100% (6/6 dairy goats and 3/3 African pygmy goats), and the cortical lesion became constant and more pronounced. By the intracerebral inoculation of pastel mink, transmissible mink encephalopathy virus was detected in the brains of several affected sheep and goats but not in extraneural sites (lymphoid tissues and intestine), except for a trace amount in the proximal colon of one goat. Even after two passages in goats, the virus remained nonpathogenic for the laboratory mouse. Despite the essential likeness of the experimental disease and scrapie, the common identity of their causal viruses remains to be determined. Even so, the results of this study are still compatible with the view that transmissible mink encephalopathy virus almost certainly is scrapie virus whose biological properties became altered by chance passage in mink, a carnivore and an aberrant host.

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.

Cerebrocortical degeneration in goats inoculated with mink-passaged scrapie virus.

Widespread spongiform degeneration of the cerebral cortex occurred in four African pygmy goats that became affected with scrapie after intracerebral inoculation with scrapie virus (Suffolk sheep brain origin) that had been passed three times in ranch mink. The occurrence of such cerebrocortical degeneration was a distinct departure from the topographic pattern of neuropathologic changes that characterizes scrapie in sheep and goats. But the cortical lesion was identical to the one found in goats that became affected with a disease otherwise indistinguishable from scrapie after intracerebral inoculation with transmissible mink encephalopathy (TME) virus that had been passed twice in mink. If TME originated from infection with wild scrapie virus, as is generally thought, then the viruses used in these two instances would be equivalent in their passage history in this aberrant host. Given this similarity, the common occurrence of the cortical lesion is thought to be consistent with the view that TME virus almost certainly is scrapie virus whose biologic properties became altered by chance passage in ranch mink.

Potent inhibition of scrapie-associated PrP accumulation by congo red.

Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using scrapie-infected neuroblastoma cells as a model system, we found that Congo red, an amyloid-binding dye, potently inhibits the accumulation of the scrapie-associated, protease-resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease-resistant PrP detected in the cultures by greater than 90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease-resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.

Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation.

The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10,000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.

Common occurrence of urethral cysts in older female ranch mink.

Cysts were found often in the proximal urethra of female sapphire and pastel mink, Mustela vison, examined at necropsy during studies on slow viral diseases. Their prevalence, the same in both color phases, was age-dependent. They occurred in less than 2% of females under 2 years old but in more than 60% of those over 7 years old. The cysts varied from ovoid vesicles 3 to 4 mm long to multilocular masses 10 to 15 mm across that greatly distended the proximal urethra and sometimes occluded it. Small cysts were not accompanied by clinical signs, but large ones often caused persistent urinary incontinence and occasionally, urine retention. The cysts contained fluid that varied from water-clear to dull yellow. They arose by expansion of small urethral glands normally present in female mink and destroyed much of the urethral wall by pressure atrophy. Although the cysts became larger and more prevalent as the mink aged, the stimulus that caused them to form was not apparent. They appear to have no counterpart in other animals.

Serological evidence for an inflammatory response in murine scrapie.

Transmissible spongiform encephalopathies (TSEs) are initiated by a novel kind of agent that produces characteristic degenerative changes in the brain without a detectable systemic inflammatory response or serological changes. A murine scrapie model was evaluated for changes in plasma concentration of serum amyloid P component (SAP), a protein that is up-regulated in infected and/or injured mice during the acute phase response (APR). C57BL10 and IRW mice inoculated with scrapie brain developed clinical scrapie 125-150 days later. At this time, concentration of plasma SAP increased in most of them. The SAP level increased > or =3-fold in >80% of the scrapie-affected C57BL10 mice and IRW male mice. A similar increase was found in <3% of respective nonscrapie control mice. The up-regulation of mouse SAP during clinical scrapie provides evidence for the activation of a systemic APR in TSE, a serological change that may be clinically useful.

Immunization of dogs with Q fever vaccines: comparison of phase I, II and phase I CMR Coxiella burnetii vaccines.

Q fever vaccines were tested in mixed breed dogs by vaccinating them with formalin-killed Coxiella burnetii whole cells (WC) in either phase I (WCI) or phase II (WCII), or the chloroform: methanol residue (CMR) subunit of phase I cells. Phase I vaccines mixed (1:1) with Freund's incomplete adjuvant (FIA) induced humoral immune responses to phases I and II antigens as measured by microagglutination assay. The CMR vaccine mixed (1:1) with FIA induced greater antigen-specific antibody levels to both phases I and II antigens than the corresponding WCI vaccine. The WCII vaccine induced antibody responses to only phase II antigens. The time course of erythema and induration after skin testing with C. burnetii antigens were suggestive of cell-mediated immunity (CMI). Although granulomas were observed with only WCI and WCII, none of the skin test antigens induced abscesses at the injection site. In contrast, axillary nodes draining the vaccine injection site developed sterile draining abscesses in all dogs by days 19 to 24 for the WCI and CMR, and day 104 for the WCII vaccines. The abscesses had resolved within 30 days after first appearance. Responses to Con A and PHA and recall antigens of lymphocytes from the blood, axillary and mesenteric nodes, and spleen at 222 days after vaccination were variable among dogs. Lymphocytes from various organs responded to one or more of the recall antigens and to both mitogens in the absence or presence of indomethacin. Although these Q fever vaccines induced humoral and CMI, either the antigens or FIA caused sterile draining abscesses.(ABSTRACT TRUNCATED AT 250 WORDS)

Congo red inhibition of scrapie agent replication.

Congo red inhibits the accumulation of protease-resistant PrP in scrapie-infected mouse neuroblastoma cells. Here we show that Congo red also inhibits the replication of scrapie infectivity in these cells. This observation is consistent with the idea that protease-resistant PrP is a vital component of the scrapie agent or that agent replication depends on the presence of protease-resistant PrP in the cell.

Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.

Information was sought on the temporal distribution of transmissible mink encephalopathy virus in royal pastel mink inoculated subcutaneously with 10(3.0) 50% intracerebral lethal doses of the Idaho strain. As determined by intracerebral assay in mink, extremely little replication of the virus occurred during the preclinical stage of infection. It seemed largely limited to lymph nodes draining the site of inoculation. Virus first appeared in the central nervous system (CNS) at 20 weeks, when all mink were still clinically normal. Early spongiform degeneration, limited to the posterior sigmoid gyrus of the frontal cortex, was first found at 28 weeks, or a few weeks before onset of clinical disease in most of the mink. Once virus reached the CNS, where greater concentrations occurred than elsewhere, it appeared in many extraneural sites (spleen, liver, kidney, intestine, mesenteric lymph node, and submandibular salivary gland). These seemingly anomalous findings, especially the limited extraneural replication of virus as a prelude to infection of the CNS, suggest that mink are not natural hosts of the virus. The results of this study support the generally held view that transmissible mink encephalopathy arises from chance or inadvertent infection of ranch mink with an exogenous virus, most likely feed-borne wild scrapie virus.

Analysis of Aleutian disease of mink parvovirus infection using strand-specific hybridization probes.

A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.