Contributions of prion strains and substrains to prion zoonotic potential and evolution

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Prions are infectious protein-only zoonotic agents that can rapidly evolve in a new replication environment. Prion strains are encoded by strain-specific conformations of PrPSc, the infectious form of the host-encoded prion protein, PrPC. Collinge and Clarke hypothesized that prion strains are a mixture of a dominant prion strain and substrains. In support of this hypothesis, we and others have shown: i) treatment of prions with anti-prion drugs can result in the emergence of a drug resistant strain, ii) physical methods (e.g. thermostability) that partially inactivate prions can select for prions with altered strain properties, and iii) strains within a given host species can have different host ranges. Despite these data, direct evidence for the existence of preexisting substrains is lacking and, consequently, the contributions of substrains to prion evolution has not been explored. The long-term goal of this work is to prevent prion evolution to a new replicative environment. The overall objective of this application is to determine the relative contributions of the dominant prion strain and substrains to interspecies transmission of prions. In this application we will test the hypothesis is that prion substrains drive prion transmission to a new PrP genotype. The hypothesis is based on our new discovery that selective reduction of PrPSc from the biologically cloned hamster-adapted drowsy (DY) strain of transmissible mink encephalopathy (TME), using two mechanistically different methods, allows for the emergence and isolation of a preexisting substrain that is distinct from DY. Moreover, this substrain unexpectedly has a different host range than DY TME. To test our hypothesis, we will first explore the frequency and diversity of preexisting substrains from stable and unstable prion strains using complimentary methods (conformational stability, protease digestion, thermostability) to selectively depopulate the dominant strain. The frequency and diversity of substrains isolated will be determined using complementary in vitro prion detection methods and animal bioassay. Second, we will explore the well-studied hamster/mouse species barrier using complementary in vitro and in vivo methods to determine the relative contributions of the dominant strain and substrains to interspecies transmission. The concept of strain interference between the dominant strain and substrains will be tested in regards interspecies transmission. These studies are significant since it is critical to understand the contributions of substrains to prion evolution as current prion treatment methods in development (vaccination, anti-prion drugs, environmental mitigation procedures) target the dominant strain. It is therefore possible that these methods will inadvertently result in the emergence of preexisting prion strains with altered zoonotic potential and/or increased pathogenicity. Overall, these studies will directly test the Collinge and Clark model of prion strain dynamics and will allow for a more precise evaluation potential of prion strains to adapt to a new replication environment (e.g., chronic wasting disease transmission to humans).