VISUALIZATION OF VIRAL CANDIDATE CDNAS IN INFECTIOUS BRAIN FRACTIONS FROM CREUTZFELDT-JAKOB-DISEASE BY REPRESENTATIONAL DIFFERENCE ANALYSIS

Creutzfeldt-Jakob Disease (CJD), a neurodegenerative and dementing dis ease of later life, is caused by a viruslike entity that is incomplete ly characterized, As in scrapie, all more purified infectious brain pr eparations contain nucleic acids. However, it has not been possible to visualize unique bands that may derive from a viral genome, We here u sed a subtractive strategy known as representational difference analys is (RDA) to uncover such sequences. To reduce the complexity of starti ng target nucleic acids, sucrose gradients were first used to select n uclease resistant particles with a defined 120S size, In CJD this sing le 120S gradient peak is highly enriched for infectivity, and contains reduced amounts of PrP (Proc. Natl. Acad. Sci, 92, 5124-8, 1995). Par allel 120S fractions from uninfected brain were made to generate subtr actor sequences. 120S particles were lysed in GdnSCN, and ng amounts o f released RNA were purified for random-primed cDNA synthesis. To capt ure representative fragments of 100 - 500 bp, cDNAs were cleaved with Mbo I for adaptor ligation and amplification. In the first experiment with moderate RDA selection, it was possible to Visualize clones from CJD cDNA that did not hybridize to control cDNA, In the second experim ent, more exhaustive subtractions yielded a discrete set of CJD derive d gel bands. Competitive hgbridization showed a subset of these bands were not present in either the control 120S cDNA or in the hamster gen ome, This represents tile first demonstration of apparently CJD-specif ic: nucleic acid bands in more purified infectious preparations. Altho ugh exhaustive cloning, sequencing and correlative titration studies n eed to be done, it is encouraging that most of the viral candidates se lected thus far have no significant homology with any previously descr ibed sequence in the database.