Evidence that homologous ssDNA is present in scrapie, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy

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.