Evidence for preexisting prion substrain diversity in a biologically cloned prion strain

Prion diseases are a group of inevitably fatal neurodegenerative disorders affecting numerous mammalian species, including Sapiens. Prions are composed of PrP(Sc), the disease specific conformation of the host encoded prion protein. Prion strains are operationally defined as a heritable phenotype of disease under controlled transmission conditions. Treatment of rodents with anti-prion drugs results in the emergence of drug-resistant prion strains and suggest that prion strains are comprised of a dominant strain and substrains. While much experimental evidence is consistent with this hypothesis, direct observation of substrains has not been observed. Here we show that replication of the dominant strain is required for suppression of a substrain. Based on this observation we reasoned that selective reduction of the dominant strain may allow for emergence of substrains. Using a combination of biochemical methods to selectively reduce drowsy (DY) PrP(Sc) from biologically-cloned DY transmissible mink encephalopathy (TME)-infected brain resulted in the emergence of strains with different properties than DY TME. The selection methods did not occur during prion formation, suggesting the substrains identified preexisted in the DY TME-infected brain. We show that DY TME is biologically stable, even under conditions of serial passage at high titer that can lead to strain breakdown. Substrains therefore can exist under conditions where the dominant strain does not allow for substrain emergence suggesting that substrains are a common feature of prions. This observation has mechanistic implications for prion strain evolution, drug resistance and interspecies transmission.