Search

Homogenized brain tissue from scrapie-infected hamsters and uninfected hamsters was subjected to subcellular fractionation to isolate unique tubulofilamentous particles termed "nemaviruses". Nucleic acid was purified from the concentrate by the phenol/chloroform extraction procedure and by alkaline gel electrophoresis; a single band of ssDNA corresponding to about 1.2 kb was visualized. The gel-purified ssDNA was mixed before inoculation with (a) MgCl2, (b) lipofectin, (c) ssDNA-binding protein and (d) normal brain homogenate. Hamsters in group b, c and d injected with a mixture of ssDNA developed the clinical disease. Brain pathology revealed generalized vacuolation, while animals injected with ssDNA mixed with MgCl2 and controls remained healthy. These results suggest that ssDNA is the genome of the scrapie agent.

Homogenized brain tissue from scrapie-infected hamsters and uninfected hamsters was subjected to sub-cellular fractionation to isolate nemavirus. Nucleic acid was extracted from these fractions, which also contained mitochondria. Agarose-gel electrophoresis revealed a band corresponding to the size of circular hamster mtDNA in both infected and uninfected samples, but slower migrating bands were observed only in samples from scrapie-infected brain. A single band of ssDNA corresponding to about 1.2 kb was purified by alkaline gel electrophoresis from the nucleic acid content of the enriched preparations of nemavirus. The ssDNA was synthesized into double-stranded DNA, cloned and sequenced. An unusual palindromic six base TACGTA repeat sequence was observed suggesting that 1.2 kb molecules consist of multiple copies of (TACGTA)n spaced along the length of the ssDNA with a preceding sequence TATATA. The comparison of the nucleotide sequence of the inserted DNA to the GenBank nucleotide database revealed no significant homology to other sequences. A probe prepared from the Nar 50 clone was hybridized against DNA prepared from scrapie, CJD, BSE and normal brains under various salt and temperature conditions. The probe reacted with a band of about 1.2 kb in scrapie, CJD and BSE but not with control normal DNA specimens, thereby confirming the presence of ssDNA in these SEs. The results suggest an intimate association between the presence of nemavirus particles and scrapie, CJD and BSE.

Homogenized normal and scrapie-infected hamster brains were subjected to subcellular fractionation. A single band of ssDNA corresponding to about 1.2 kb was purified by alkaline gel electrophoresis from the nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles. The ssDNA was synthesized into double-stranded DNA using Taq polymerase with four dNTP for extension. The cDNA synthesized was inserted in M13mp10, cloned and sequenced. An unusual palindromic six-base TACGTA repeat sequence was obtained and confirmed by an independent automated pathway and by cutting with a specific restriction enzyme. Comparison of the nucleotide sequence of the inserted DNA with the GenBank nucleotide database revealed no significant homology with those sequences. A probe prepared from the Nar 50 clone hybridized with the scrapie DNA band of about 1.2 kb noted above; however, no hybridization was observed with normal DNA, thus confirming the presence of ssDNA in scrapie. The presence of palindromic sequences in the scrapie genome could explain why many previous searches have revealed no evidence for a scrapie-specific nucleic acid.

Examination of thin sections from the cerebral cortex of scrapie-infected hamster brains revealed characteristic circular 26-30 nm diameter tubulofilamentous particles, identical to those previously described in both experimentally induced scrapie in mice, hamsters and natural scrapie of sheep, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease and mice and chimpanzees infected with Creutzfeldt-Jakob disease. Longitudinal forms of tubulofilamentous particles were also observed in dendrites and myelinated axons. Both transverse and longitudinally cut particles were readily distinguished from microtubules and synaptic vesicles, thus there appears to be no relationship between tubulofilamentous particles, and microtubules or synaptic vesicles.

We observed a marked increase in multimeric mitochondrial DNA (mtDNA) in brains of scrapie-infected hamsters compared with those of uninfected hamsters. Homogenized brain tissue was subjected to subcellular fractionation to isolate scrapie-associated fibrils and tubulofilamentous structures. Nucleic acids were extracted from the scrapie-associated fibril/tubulofilament fraction which also contained mitochondria. Agarose gel electrophoresis revealed a band corresponding to the size of circular hamster mtDNA in both infected and uninfected samples, but slower migrating bands were observed only in samples from scrapie-infected brain. We showed by molecular cloning, nucleotide sequencing, and Southern blotting that the slower migrating bands are mtDNA. These findings confirm the recent demonstration by differential hybridization that multimeric mtDNA occurs in hamster scrapie brain. Elevated levels of multimeric circular mtDNA have been reported previously in various tumors and cultured cell lines.

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.

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 X 10(6) daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.

A search procedure at the ultrastructural level revealed characteristic particulate structures in mice of two further laboratory stocks experimentally inoculated with scrapie. The structures represent a specific change related to scrapie disease; their relationship to the scrapie agent is discussed.

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of filamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubulofilamentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.

The development of scrapie-associated particles and lesions in four regions of the brain was studied in mice over a period of 30 weeks. Characteristic tubulovesicular particles, identical to those previously described, were first found about half way through the incubation period in mice inoculated by four different routes. The particles are found in brains with scrapie and other spongiform encephalopathies; they have never been seen in other conditions, and potentially represent the infectious agent.

Abnormal tubulovesicular particles of an average diameter of 23 nm have been observed in brains of mice with scrapie as well as in other animals with spongiform encephalopathies, but they were thought to be absent from the brains of hamsters with scrapie in which the highest known concentrations of the infectious agent occur. We observed in neuronal processes of hamsters as well as mice clusters of those tubulovesicular structures, most often in postsynaptic terminals. Such particles have now been seen regularly in both experimental and natural scrapie in all species examined as well as in other spongiform encephalopathies.

A simple method was devised for negative-stain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected hamsters contained large masses of tubulofilamentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubulofilaments were found in stages of fragmentation, an outer coat appearing t0 strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.