Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Journal of Visualized Experiments
By: , and 

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Abstract

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Additional publication details

Publication type Article
Publication Subtype Journal Article
Title Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay
Series title Journal of Visualized Experiments
DOI 10.3791/52522
Volume 97
Year Published 2015
Language English
Publisher JoVE
Contributing office(s) National Wildlife Health Center
Description e52522
Online Only (Y/N) N
Additional Online Files (Y/N) N