Characterization of unconventional agent (scrapie agent): analysis by dissociation and reconstitution of SAF fraction and trial study for detection of scrapie specific nucleic acid in the fraction.

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In order to characterize scrapie agent, following experiments were carried out on SAF which associate with scrapie infectivity, consist of a host protein, PrP, and have been prepared from brain. 1) Effect of antiserum to SAF was examined for incubation period of scrapie in mice. The incubation period did change by treatment with the antiserum. 2) To examine whether the association of SAF and the agent was mere adsorption of the agent to SAF, detection of PrP in slpeen and lymph node at various time after infection, in which considerable level of scrapie infectivity exists from early stage of infection, was carried out. PrP was detected in these organs from preclinical stages indicating that association of the infectivity and SAF was intimate.Moreover, the results suggest that detection PrP can be applied for scrapie diagnosis in preclinical stage. 3) Since we found that SAF dissolves slightly in butanol, butanol solution of ASF was fractionated by column chromatography. From the fracti … More ons containing PrP, butanol was removed by mixing with water. Fine fibrils were observed by an electron microscope in the fractions, indicating that SAF was partially reconstituted. Mice inoculated with unfractionated butanol sample showed scrapie signs but with the fractions within one year and they were under observation. 4) It has been thought that PrP self-assembles and forms SAF during purification procedure including detergent treatment of brain membrane. To prepare soluble PrP fraction without exposure to detergent, urea was used to dissolve the membrane, and fractionated by glycerol gradient centrifugation. PrP was detected even in fractions consisting with 50% glycerol, and PrP in such fractions formed large aggregates showing birefringence. Fibrils were dissociated from the aggregates by brief exposure to a detergent, indicating that PrP exists in scrapie brain as aggregates, and SAF is purified from the dissociated aggregates by detergent treatment. 5) To detect infectious nucleic acid in SAF enriched fraction, a fraction containing nucleic acids was prepared from protease treated SAF fraction by phenol extraction. The fraction was introduced in to mouse cultured cells by the calcium phosphate-precipitation method. The cells were inoculated in to mice after 2 weeks. No mice inoculated showed scrapie signs within their life span. Less