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Updated: May 27, 2026
Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay
Published on: March 10, 2015
Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions
Mikael Klingeborn1, Brent Race, Kimberly D Meade-White
1Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA.
Abstract:
Prions are unconventional infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases, or prion diseases. The biochemical nature of the prion infectious agent remains unclear. Previously, using a protein misfolding cyclic amplification (PMCA) reaction, infectivity and disease-associated protease-resistant prion protein (PrPres) were both generated under cell-free conditions, which supported a nonviral hypothesis for the agent. However, these studies lacked comparative quantitation of both infectivity titers and PrPres, which is important both for biological comparison with in vivo-derived infectivity and for excluding contamination to explain the results. Here during four to eight rounds of PMCA, end-point dilution titrations detected a >320-fold increase in infectivity versus that in controls. These results provide strong support for the hypothesis that the agent of prion infectivity is not a virus. PMCA-generated samples caused the same clinical disease and neuropathology with the same rapid incubation period as the input brain-derived scrapie samples, providing no evidence for generation of a new strain in PMCA. However, the ratio of the infectivity titer to the amount of PrPres (specific infectivity) was much lower in PMCA versus brain-derived samples, suggesting the possibility that a substantial portion of PrPres generated in PMCA might be noninfectious.
Insights
Prion diseases are caused by infectious agents called prions. Cell-free amplification of prions in vitro supports a nonviral hypothesis, with increased infectivity and disease characteristics matching natural prion infections.
Area of Science:
- Neuroscience
- Biochemistry
- Infectious Diseases
Background:
- Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathies (TSEs).
- The precise biochemical nature of the prion infectious agent remains incompletely understood.
- Previous cell-free protein misfolding cyclic amplification (PMCA) studies generated infectivity and protease-resistant prion protein (PrPres) but lacked comparative quantitation.
Purpose of the Study:
- To quantitatively compare infectivity titers and PrPres levels generated through cell-free PMCA with in vivo-derived infectivity.
- To provide robust evidence supporting or refuting the nonviral hypothesis for prion infectivity.
- To assess whether PMCA-generated prions induce the same disease characteristics as naturally occurring prion diseases.
Main Methods:
- Utilized protein misfolding cyclic amplification (PMCA) over four to eight rounds.
- Performed end-point dilution titrations to quantify infectivity titers in both PMCA-generated and control samples.
- Compared clinical disease, neuropathology, and incubation periods of PMCA-generated samples with brain-derived scrapie samples.
Main Results:
- Demonstrated a greater than 320-fold increase in infectivity in PMCA-generated samples compared to controls.
- PMCA-generated prions induced identical clinical disease, neuropathology, and incubation periods as input scrapie samples, indicating no new strain generation.
- Observed a significantly lower ratio of infectivity titer to PrPres amount (specific infectivity) in PMCA samples versus brain-derived samples.
Conclusions:
- The substantial increase in cell-free infectivity strongly supports the hypothesis that the prion agent is not a virus.
- PMCA successfully replicates prion infectivity and disease-causing properties without evidence of new strain formation.
- A portion of the PrPres generated during PMCA may be noninfectious, as indicated by the reduced specific infectivity.
