Species barriers for chronic wasting disease by in vitro conversion of prion protein Li Li a , Michael B. Coulthart b , Aru Balachandran c , Avi Chakrabartty d , Neil R. Cashman a, * a Brain Research Centre, Division of Neurology, Department of Medicine, University of British Columbia and Vancouver Coastal Health, UBC Hospital, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 b Prion Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Man., Canada R3E 3R2 c National Reference Laboratory for Scrapie and CWD, Animal Diseases Research Institute, Canadian Food Inspection Agency, 3851 Fallowfield Road, Nepean, Ont., Canada K2H 8P9 d University Health Network, Department of Medical Biophysics, University of Toronto, Toronto, Ont., Canada M5G 1L7 Received 6 October 2007 Available online 25 October 2007 Abstract Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP CWD of elk can readily induce the conversion of normal cervid PrP (PrP C ) molecules to a protease-resistant form, but is less efficient in converting the PrP C of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP CWD -induced conversion can be greatly enhanced in all species. Our results dem- onstrate that PrP C from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP C can apparently overcome the structural barriers between more distant species. Ó 2007 Elsevier Inc. All rights reserved. Keywords: CWD; PrP C ; PrP Sc ; In vitro conversion; Species barrier Chronic wasting disease (CWD) is a cervid form of transmissible spongiform encephalopathy (TSE) or prion disease. CWD’s rapid spread from’ Colorado to other states [1,2], to Canadian provinces (Alberta, Saskatche- wan) [1] and to Korea [2,3] has raised concerns about its species tropism [4–6]. CWD has been transmitted to cattle via intracerebral inoculation [7], and to other animals, including ferrets, mink, and goats [8,9]. Reports document- ing CWD prions in the muscle [10,11], blood, and saliva [12] of infected cervids, have heightened interest in the dis- ease by public health agencies [13]. CWD and other TSEs are believed to be due to the tem- plate-directed accumulation of disease-associated prion protein, generically designated PrP Sc . PrP C in brain homogenates can be converted to a protease-resistant form by incubation with PrP Sc ‘‘seeds’’ which are thought to recapitulate the template-directed misfolding of prion pro- tein in disease [14,15], including protein misfolding cyclic amplification (PMCA) [15]. We have previously reported that partially denatured human brain PrP C (which may mimic a PrP conversion intermediate [16]) is a superior sub- strate for templated in vitro conversion compared with untreated PrP C in an incubation-shaking assay that does not utilize PMCA sonication [17]. Materials and methods Reagents and antibodies. Proteinase K (PK) was purchased from Invitrogen. Mouse monoclonal antibody 6H4 was from Prionics Co. (Zu ¨ rich, Switzerland). Horseradish peroxidase-conjugated sheep anti- 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.087 * Corresponding author. Fax: +1 604 822 7299. E-mail address: Neil.Cashman@vch.ca (N.R. Cashman). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 364 (2007) 796–800