Summary Prions are unconventional infectious agents composed exclusively by the misfolded prion protein (PrPSc), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrPC). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here we report the generation of novel infectious prions by inter-species transmission of PrPSc misfolding in vitro. Hamster PrPC misfolded by mixing with mouse PrPSc generated new prions that were infectious to wild type hamsters. Similarly, new mouse prions were generated by crossing the species barrier in the opposite direction. A detailed characterization of the infectious, biochemical and histological properties of the disease produced indicate that the in vitro generated material across the species barrier correspond to new prion strains. Successive rounds of PMCA amplification result in a progressive adaptation of the in vitro produced prions, in a process reminiscent to the strain stabilization process observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool to investigate cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrPSc). Disease is transmitted by the autocatalytic propagation of PrPSc misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrPSc generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrPSc caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrPSc properties.This research was supported in part by NIH grants NS0549173 and AG014359 to CS

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