NR AXQP
AU Kurt,T.D.; Perrott,M.R.; Wilusz,C.J.; Wilusz,J.; Supattapone,S.; Telling,G.C.; Zabel,M.D.; Hoover,E.A.
TI Efficient in Vitro Conversion of CWD PrP-RES by Serial Protein Misfolding Cyclic Amplification
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Oral Abstracts FC1.4
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Vortrag
AB
Purpose: Chronic wasting disease (CWD) of cervids is a transmissible spongiform encephalopathy (TSE) of increasing prevalence in North America. Infectious CWD prions have been demonstrated in saliva and blood of deer by bioassay, however, detection of PrPres in these fluids has not been possible using conventional in vitro assays. To enhance CWD PrPres detection and study potential trans-species infection, we have employed both non-denaturing amplification (Supattapone et al.) and proteinmisfolding cyclic amplification (PMCA)(Soto et al.) to convert and amplify cervid (and other species) PrPc to CWD PrPres in vitro.
Materials/method: PrPc from brain tissue of cervids and other species was converted to protease-resistant PrPres by spiking normal brain homogenates (NBH) (10% w/v) with CWD-positive deer brain homogenate followed by incubation at 37C with 40 sec sonic pulses delivered every 30 min over a period of 48 hours. Serial PMCA was accomplished by transfer of aliquots of each reaction mixture into fresh NBH followed by re-amplification. CWD PrPres was detected by immunoblotting after proteinase K digestion of control and test samples.
Results: Using NBH from cervid PrP 1536 transgenic mice (Browning et al.) in serial PMCA, we have succeeded in amplifying CWD PrPres over 6.5 x 109-fold after six rounds. Surprisingly, PMCA using white-tailed deer NBH resulted in just ~10-fold increases in PrPres per round, a vexingly low yield given the efficient transmission of CWD in nature. That Tg(cerPrP)1536 NBH contains ~4 fold greater concentration of PrPc compared with deer NBH may provide at least a partial explanation for this difference. Finally, in a series of trans-species PrPcWD amplification experiments, we have documented reasonably efficient PrPcWD amplification using NBH from ferrets, a species shown to be susceptible to CWD infection in vivo.
Conclusion: We report (to our knowledge for the first time) efficient CWD PrPres amplification in vitro. We are currently applying this methodology to detection of PrPcWD in body fluids and excreta from infected animals as well as to probe potential susceptibility of non-cervids to CWD infection.
AD T. Kurt, M.R. Perrott, C.J. Wilusz, J. Wilusz, M.D. Zabel, E.A. Hoover, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Department of Microbiology, Immunology & Pathology, USA; S. Supattapone, Dartmouth Medical School, Department of Biochemistry, USA; G.C. Telling, University of Kentucky, Department of Molecular Biology and Genetics, USA
SP englisch
PO Schottland