Crossing the Species Barrier by PrPSc Replication In Vitro Generates Unique Infectious Prions

Abstract

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.

Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions

“…However, under other conditions where higher amounts of input seed and increased cycles per round were used, PMCA conversion even across a resistant species barrier has been reported (28). In our experiments using conditions similar to those previously described (21), no significant differences in clinical signs, incubation periods, and histopathological or biochemical data were found between animals inoculated with brain-derived input seed or PMCA-derived products from rounds 4, 6, and 8.…”

“…As opposed to earlier studies where prion infectivity was often assayed in material from a single round after >15 serial rounds of PMCA (21-24), we analyzed multiple early rounds of several different hamster and transgenic mouse PMCA experiments. Furthermore, to estimate infectivity titers all but one (22) of these earlier studies used an incubation time assay based on a standard curve using brain-derived prion infectivity (25), but such a standard curve may not be valid for PMCA-derived infectivity (26,27), especially if a new prion strain is generated (28). In contrast, in the present experiments the use of end-point dilution titrations enabled us to follow the disappearance of the input infectivity in control reactions and accurately titer any newly generated infectivity.…”

Prion replication without host adaptation during interspecies transmissions

“…In serial PMCA, prions replicate indefinitely through successive rounds of dilutions and amplification, and this strategy has been used to traverse species barriers and generate species-adapted prions (35). Because sPMCA generated infectious rabbit prions, a species that was considered resistant to prion disease (36), we used the same approach to produce horse prions.…”

Protein Misfolding Cyclic Amplification of Prions

“…The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP . This technique has proven to effectively recapitulate the species and strain specificity of PrP Sc conversion from PrP C , to emulate prion strain interference, and to amplify very low levels of PrP Sc from infected tissues, fluids, and environmental samples 6,7,16,23 . This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results.…”