Ultra-Efficient PrPSc Amplification Highlights Potentialities and Pitfalls of PMCA Technology

PLoS Pathogens

Joaquin Castilla¹  Michele Angelo Di Bari²  Romolo Nonno²  Cecilia Bucalossi³  Umberto Agrimi³  Gabriele Vaccari³  Gian Mario Cosseddu³  Natalia Fernandez-Borges³ 

[1] 33458CIC bioGUNE, Parque Tecnológico de Bizkaia, Derio, Spain;Department of Infectology, The SCRIPPS Research Institute, Jupiter, Florida, United States of America;Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

关键词: Voles;    Prion diseases;    Animal prion diseases;    Sonication;    Pipettes;    Scrapie;    Creutzfeldt-Jakob disease;    Sheep;   

DOI  :  10.1371/journal.ppat.1002370

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrPSc in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrPSc in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrPSc in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrPSc was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrPSc to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.

【 授权许可】

CC BY