title: Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay.
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 vitro prion 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.
authors: USGS National Wildlife Health Center; cjjohnson@usgs.gov.
authors: Christopher J
authors: CJ
authors: Johnson
authors: Department of Soil Science, University of Wisconsin-Madison.
authors: Christina M
authors: CM
authors: Carlson
authors: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
authors: Aaron R
authors: AR
authors: Morawski
authors: Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin-Madison.
authors: Alyson
authors: A
authors: Manthei
authors: Department of Neurology, University of British Columbia.
authors: Neil R
authors: NR
authors: Cashman
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D006224:Cricetinae; D003670:Deer; D046008:Lynx; D051379:Mice; D017096:Prion Diseases / Q000378:metabolism / Q000635:transmission*; D011328:Prions / Q000378:metabolism*; D013045:Species Specificity
chemical_list: D011328:Prions