Friday, May 14, 2010

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Published Online May 13, 2010 Science DOI: 10.1126/science.1187107 Science Express Index


Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Rachel C. Angers,1,* Hae-Eun Kang,2 Dana Napier,2 Shawn Browning,1, Tanya Seward,2 Candace Mathiason,4 Aru Balachandran,5 Debbie McKenzie,6 Joaquín Castilla,7 Claudio Soto,8 Jean Jewell,9 Catherine Graham,10 Edward A. Hoover,4 Glenn C. Telling1,2,3,

Prions are infectious proteins composed of PrPSc, which induces conformational conversion of host-encoded PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties, but comprised of PrPSc with indistinguishable biochemical characteristics. While CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk PrP differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.


The identification and characterization here of distinct CWD strains with similar conformations, and the influence of PrP primary structure on their stabilities, is of importance when considering the potential for transmission to species outside the family cervidae. While CWD prions have reassuringly failed to induce disease in transgenic mice expressing human PrP (10, 30), because of the risk of prion exposure from contaminated venison (13) and other infected materials (14), systematically addressing the tissue distributions of CWD1 and CWD2 and their zoonotic potentials would appear to be high priorities.

(10) Transmission of Elk and Deer Prions to Transgenic Mice

(30) Neurobiology of Disease Chronic Wasting Disease of Elk: Transmissibility to Humans Examined by Transgenic Mouse Models

(13) Prions in Skeletal Muscles of Deer with Chronic Wasting Disease;311/5764/1117

(14) Volume 15, Number 5–May 2009 Research Chronic Wasting Disease Prions in Elk Antler Velvet

1 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, Lexington, KY 40536, USA. 2 Sanders Brown Center on Aging, University of Kentucky Medical Center, Lexington, KY 40536, USA. 3 Department of Neurology, University of Kentucky Medical Center, Lexington, KY 40536, USA. 4 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5 Canadian Food Inspection Agency, Ottawa, Ontario, K2H 8P9, Canada. 6 Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada. 7 CIC bioGUNE & IKERBASQUE, Basque Foundation for Science, 48992 Derio & 48011 Bilbao, Bizkaia, Spain. 8 University of Texas Medical School at Houston, Houston, TX 77030, USA. 9 Department of Veterinary Sciences, University of Wyoming, Laramie, WY, USA. 10 Canadian Food Inspection Agency, Lethbridge, Alberta, T1J 3Z4, Canada. * Present address: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Present address: Department of Infectology, Scripps Research Institute, Jupiter, FL, USA.

To whom correspondence should be addressed. E-mail:


Received for publication 14 January 2010. Accepted for publication 1 April 2010.

Supporting Online Material for

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Rachel C. Angers, Hae-Eun Kang, Dana Napier, Shawn Browning, Tanya Seward, Candace Mathiason, Aru Balachandran, Debbie McKenzie, Joaquín Castilla, Claudio Soto, Jean Jewell, Catherine Graham, Edward A. Hoover, Glenn C. Telling*

*To whom correspondence should be addressed. E-mail:

Published 13 May 2010 on Science Express DOI: 10.1126/science.1187107

This PDF file includes: Materials and Methods Table S1 References


Supporting Online Material

Materials and Methods

Transgenic mice and inocula Transgenic mice expressing deer or elk PrP coding sequences, referred to as Tg(CerPrP)1536+/- and Tg(CerPrP-E226)5037+/- respectively, have been described previously (S1, S2). All transmitted isolates in this study originated from deer and elk expressing wild type PRNP coding sequences. The 03W1755 elk used in PMCA studies was heterozygous (M/L) at codon 132. Ten % (w/v) homogenates, in phosphate buffered saline (PBS) lacking calcium and magnesium ions, of cervid and mouse brains were prepared by repeated extrusion through an 18 gauge followed by a 21 gauge syringe needle.

Determination of Incubation Periods Groups of anesthetized mice were inoculated intracerebrally with 30 µl of 1 % (w/v) brain extracts prepared and diluted in PBS, or 1 % v/v of the final PMCA product diluted in PBS. Groups of mice were monitored thrice weekly for the development of prion disease. Following a relatively non-specific prodromal phase, early definitive and progressive clinical signs included stimulation-induced hyperexcitability, and flattened posture, culminating in profound ataxia toward the endpoint of disease. CWD-affected mice were rarely kyphotic and maintained a deep pain reflex at end stage. Inoculated mice were diagnosed with prion disease following the progressive development of at least three clinical signs, the time from inoculation to the onset of definitive and subsequently progressive clinical signs being referred to as the incubation time.


Analysis of PrP Animals whose death was obviously imminent were euthanized and their brains taken for biochemical and histopathological studies. For PrP analysis in brain extracts, total protein content from 10 % brain homogenates prepared in PBS was determined by bicinchoninic acid assay (Pierce Biotechnology Inc., Rockford, IL). Brain extracts were either untreated or treated with 40 µg/ml PK for one hour at 37oC in the presence of 2 % sarkosyl and the reaction was terminated with 4 mM phenyl methyl sulfonyl fluoride. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrophoretically transferred to PVDF-FL membranes (Millipore, Billerica, MA), which were probed with anti-PrP mAbs followed by horse radish peroxidaseconjugated sheep anti-mouse IgG, developed using ECL-plus detection (Amersham), and analyzed using an FLA-5000 scanner (Fuji). To determine the relative values of CerPrPSc glycoforms, band intensities were analyzed by densitometry of Western blots using the FLA-5000 scanner.

For histoblot analysis, mice exhibiting neurological dysfunction were humanely killed and their brains immediately frozen on dry ice. Ten µm thick cryostat sections were transferred to nitrocellulose as previously described (S3). Histoblots were immunostained with mAb 6H4 followed by alkaline phosphataseconjugated sheep anti-mouse secondary antibody. Images were captured with a Nikon SMZ1000 microscope with Photometrics Coolsnap CF digital imager and processed with MetaMorph software.

PrPSc in brain homogenates of terminally sick mice was also analyzed by conformational stability assay (S4-S7). The relative amounts of bands


representing PK resistant CerPrPSc were analyzed by densitometry of Western blots using the FLA-5000 scanner. The sigmoidal dose-response was plotted using a four-parameter algorithm and non-linear least square fit. The Gdn.HCl concentration required to denature 50% of CerPrPSc is denoted as the (Gdn.HCl)1/2 value. For histopathological stu dies, brains were dissected rapidly after sacrifice of the animal and immersion fixed in 10% buffered formalin. Tissues were embedded in paraffin and 8 µm thick coronal microtome sections were mounted onto positively charged glass slides. Analysis of PrP in the brains of mice by IHC was performed as previously described (S8) using anti-PrP mAb 6H4 as primary antibody, and IgG1 biotinylated goat anti-mouse secondary antibody (Southern Biotech). Following inactivation of endogenous peroxidases by incubation in 3% H2O2 in methanol, peroxidase immunohistochemistry was used to evaluate the extent of reactive astrocytic gliosis using antibodies to glial fibrillary acidic protein. Detection was with Vectastain ABC reagents and slides were developed with diaminobenzidine. Digitized images for figures were obtained by light microscopy using a Nikon Eclipse E600 microscope equipped with a Nikon DMX 1200F digital camera.

Neuropathological Lesion Profiling Paraffin-embedded mouse brains were sectioned coronally to areas corresponding to the five levels of the brain that contained the mouse brain regions of interest. Brain sections were stained with hematoxylin and eosin. Images of the each of the brain regions were captured using a Photometrics Cool Snap digital camera and a Nikon Eclipse E600


microscope. The extent of vacuolar degeneration in the cerebral grey matter was assessed using a semi-quantitative method for discriminating prion strains (S9). The numbers of vacuoles per field were manually counted. Text In addition to intrinsic strain characteristics, time to onset of disease is dependent on prion titers (S10). Both effects were evident in these studies. Preparations containing low CWD prions titers produce longer incubation times in Tg(CerPrP)1536+/- mice than higher titer isolates (S2). Prolonged incubation times resulting from primary transmission of low titer CWD isolates generally shorten on second passage in syngeneic hosts, while strain-related incubation time properties are expected to persist during serial transmission. To begin to distinguish the effects of strain and titer on the variable incubation times observed during primary transmissions, we performed serial transmissions in Tg(CerPrP)1536+/- mice (Table S1). Titer-related reduction in mean incubation time was a feature of many of serially passaged isolates (Table S1). Generally, on second passage, there was less variance of incubation times for both strains, an effect that was also likely to be related to more consistent prion titers. Factors affecting CWD prion titers include the stage of disease in affected deer and elk, the neuroanotomical locations from which prions were isolated, and possible effects of post-mortem interval.

The most extreme effects of titer were observed during transmission of the 012-22012 elk isolate. Despite a 387 d incubation time, CWD1 neuropathology


was registered in the only mouse available for analysis following transmission of elk isolate 012-22012 (Fig. 1A); 3 of 8 inoculated mice in this cohort did not develop disease (Table S1 and Fig. 1A). The protracted time to onset of disease and the less than 100 % attack rate on primary passage suggests that the titer of CWD1 prions in this elk isolate was close to the endpoint of sensitivity of the bioassay. Consistent with this notion, serial passage of 012-22012 prions from the brain of a second diseased mouse with a 380 d incubation time, produced a rapid mean incubation time of 208 ± 4 d in 8 inoculated mice and CWD1 neuropathology in all analyzed mice (n = 5) (Fig. 2A and Table S1).


Table S1: Transmission of CWD prions to Tg(CerPrP)1536+/- mice Incubation time, mean days ± SD (n/n0)

Inoculum Origin First Second


012-22012 Colorado 384 ± 3.3 (5/8) 208 ± 3.5 (8/8) 012-09442 Colorado 208 ± 16.9 (8/8) 307 ± 25.3 (6/6) 02-0306 Saskatchewan 225 ± 8.3 (7/7) 238 ± 38.1 (7/7) 12389 Wyoming 230 ± 24.4 (8/8) 001-44720 Colorado 231 ± 13.7 (7/7) 248 ± 37.5 (8/8) 7378-47 Wyoming 235 ± 5.5 (8/8) 230 ± 28.6 (7/7) 001-403022 Colorado 271 ± 35.9 (8/8) 235 ± 38.1 (8/8) 04-0306 Saskatchewan 281 ± 14.9 (7/7) 211 ± 7.5 (7/7) CWD pool Alberta 293 ± 30.9 (6/6) 01-0306 Saskatchewan 322 ± 25.3 (8/8) 274 ± 29.3 (8/8) 03-0306 Saskatchewan 335 ± 12.6 (7/7) 226 ± 45.9 (9/9)

Mule deer

8481 Wyoming 173 ± 3.8 (7/7) 217 ± 28.8 (7/7) 978-24384 Colorado 211 ± 22.5 (7/7) 229 ± 26.8 (5/5) D10 Colorado 228 ± 28.9 (15/15) 217 ± 28.3 (8/8) D92 Colorado 232 ± 48.8 (15/15) 244 ± 46.6 (7/7) 9179 Wyoming 239 ± 64.6 (7/7) 216 ± 32.0 (7/7) 989-09147 Colorado 250 ± 6.5 (8/8) 325 ± 36.0 (5/5) W97 Colorado 254 ± 27.1 (5/5) 226 ± 43.9 (7/7) 8905 Wyoming 259 ± 63.3 (8/8) 238 ± 28.6 (8/8) Db99 Colorado 259 ± 11.2 (7/7) 246 ± 15.8 (4/4) 7138 Wyoming 260 ± 46.6 (7/7) 216 ± 22.9 (7/7) CWD Pool Colorado 264 ± 9.3 (7/7) 207 ± 6.0 (6/6) 33968 Colorado 278 ± 27.0 (6/6) 239 ± 19.5 (8/8) H92 Colorado 283 ± 20.4 (6/6) 259 ± 44.6 (8/8) 04-22412 Wyoming 284 ± 54.4 (6/6) 001-39647 Colorado 289 ± 7.9 (5/5) 217 ± 47.1 (8/8) V92 Colorado 310 ± 30.5 (7/7) 288 ± 16.0 (8/8)

Whitetail deer

Wisconsin 200 ± 19.2 (6/6)2 206 ± 3.7 (8/8)


Elk 03W1755 Wyoming/Texas3 446 ± 22.6 (5/5)

Deer 04-22412 Wyoming/Texas3 264 ± 73.1 (6/6)

Total CWD14 212 ± 34.1 (n = 64) 206 ± 11.9 (n = 68)

Total CWD24 306 ± 48.5 (n = 78) 286 ± 22.1 (n = 46)

Saline 410 – 597 (0/7)

None 421 – 490 (0/7)

1 The number of mice developing prion disease (n), divided by the number inoculated (n0) is shown in parentheses. Mice dying of causes unrelated to prion disease were excluded.

2 PrPSc in this sample was precipitated with sodium phosphotungstate prior to inoculation.


3 Samples originated from Wyoming elk and deer; PMCA was accomplished in Texas.

4 Mean incubation times for primary transmission of naturally-occurring and PMCAgenerated CWD prions were determined in 64 neuropathologically confirmed mice with the CWD1 pattern, and 78 neuropathologically confirmed mice with the CWD2 pattern. Mean incubation times for secondary transmissions of CWD prions were determined in 68 neuropathologically confirmed mice with the CWD1 pattern, and 46 neuropathologically confirmed mice with the CWD2 pattern. For both primary and secondary passages, incubation times of CWD1 and CWD were different (p



Chad Johnson1, Judd Aiken2,3,4 and Debbie McKenzie4,5 1 Department of Comparative Biosciences, University of Wisconsin, Madison WI, USA 53706 2 Department of Agriculture, Food and Nutritional Sciences, 3 Alberta Veterinary Research Institute, 4.Center for Prions and Protein Folding Diseases, 5 Department of Biological Sciences, University of Alberta, Edmonton AB, Canada T6G 2P5

The identification and characterization of prion strains is increasingly important for the diagnosis and biological definition of these infectious pathogens. Although well-established in scrapie and, more recently, in BSE, comparatively little is known about the possibility of prion strains in chronic wasting disease (CWD), a disease affecting free ranging and captive cervids, primarily in North America. We have identified prion protein variants in the white-tailed deer population and demonstrated that Prnp genotype affects the susceptibility/disease progression of white-tailed deer to CWD agent. The existence of cervid prion protein variants raises the likelihood of distinct CWD strains. Small rodent models are a useful means of identifying prion strains. We intracerebrally inoculated hamsters with brain homogenates and phosphotungstate concentrated preparations from CWD positive hunter-harvested (Wisconsin CWD endemic area) and experimentally infected deer of known Prnp genotypes. These transmission studies resulted in clinical presentation in primary passage of concentrated CWD prions. Subclinical infection was established with the other primary passages based on the detection of PrPCWD in the brains of hamsters and the successful disease transmission upon second passage. Second and third passage data, when compared to transmission studies using different CWD inocula (Raymond et al., 2007) indicate that the CWD agent present in the Wisconsin white-tailed deer population is different than the strain(s) present in elk, mule-deer and white-tailed deer from the western United States endemic region.

Sunday, April 12, 2009

CWD UPDATE Infection Studies in Two Species of Non-Human Primates and one Environmental reservoir infectivity study and evidence of two strains

Thursday, April 03, 2008

A prion disease of cervids: Chronic wasting disease

2008 1: Vet Res. 2008 Apr 3;39(4):41

A prion disease of cervids: Chronic wasting disease

Sigurdson CJ.


*** twenty-seven CJD patients who regularly consumed venison were reported to the Surveillance Center***,


full text ;

From: TSS (


Date: September 30, 2002 at 7:06 am PST

From: "Belay, Ermias"


Cc: "Race, Richard (NIH)" ; ; "Belay,


Sent: Monday, September 30, 2002 9:22 AM


Dear Sir/Madam,

In the Archives of Neurology you quoted (the abstract of which was attached to your email), we did not say CWD in humans will present like variant CJD.

That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091).

Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.

Ermias Belay, M.D.

Centers for Disease Control and Prevention

-----Original Message-----


Sent: Sunday, September 29, 2002 10:15 AM

To:;; ebb8@CDC.GOV



Sunday, November 10, 2002 6:26 PM ......snip........end..............TSS

SEE also ;

A. Aguzzi - Chronic Wasting Disease (CWD) also needs to be addressed. Most serious because of rapid horizontal spread and higher prevalence than BSE in UK, up to 15% in some populations. Also may be a risk to humans - evidence that it is not dangerous to humans is thin.


Chronic Wasting Disease and Potential Transmission to Humans

Ermias D. Belay,* Ryan A. Maddox,* Elizabeth S. Williams,? Michael W. Miller,? Pierluigi Gambetti,§ and Lawrence B. Schonberger*

*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; ?University of Wyoming, Laramie, Wyoming, USA; ?Colorado Division of Wildlife, Fort Collins, Colorado, USA; and §Case Western Reserve University, Cleveland, Ohio, USA

Suggested citation for this article: Belay ED, Maddox RA, Williams ES, Miller MW, Gambetti P, Schonberger LB. Chronic wasting disease and potential transmission to humans. Emerg Infect Dis [serial on the Internet]. 2004 Jun [date cited]. Available from:


Chronic wasting disease (CWD) of deer and elk is endemic in a tri-corner area of Colorado, Wyoming, and Nebraska, and new foci of CWD have been detected in other parts of the United States. Although detection in some areas may be related to increased surveillance, introduction of CWD due to translocation or natural migration of animals may account for some new foci of infection. Increasing spread of CWD has raised concerns about the potential for increasing human exposure to the CWD agent. The foodborne transmission of bovine spongiform encephalopathy to humans indicates that the species barrier may not completely protect humans from animal prion diseases. Conversion of human prion protein by CWD-associated prions has been demonstrated in an in vitro cell-free experiment, but limited investigations have not identified strong evidence for CWD transmission to humans. More epidemiologic and laboratory studies are needed to monitor the possibility of such transmissions.

snip...full text ;

Volume 12, Number 10-October 2006


Human Prion Disease and Relative Risk Associated with Chronic Wasting Disease

Samantha MaWhinney,* W. John Pape,? Jeri E. Forster,* C. Alan Anderson,?§ Patrick Bosque,?¶ and Michael W. Miller#

*University of Colorado at Denver and Health Sciences Center, Denver, Colorado, USA; ?Colorado Department of Public Health and Environment, Denver, Colorado, USA; ?University of Colorado School of Medicine, Denver, Colorado, USA; §Denver Veteran's Affairs Medical Center, Denver, Colorado, USA; ¶Denver Health Medical Center, Denver, Colorado, USA; and #Colorado Division of Wildlife, Fort Collins, Colorado, USA

Suggested citation for this article

The transmission of the prion disease bovine spongiform encephalopathy (BSE) to humans raises concern about chronic wasting disease (CWD), a prion disease of deer and elk. In 7 Colorado counties with high CWD prevalence, 75% of state hunting licenses are issued locally, which suggests that residents consume most regionally harvested game. We used Colorado death certificate data from 1979 through 2001 to evaluate rates of death from the human prion disease Creutzfeldt-Jakob disease (CJD). The relative risk (RR) of CJD for CWD-endemic county residents was not significantly increased (RR 0.81, 95% confidence interval [CI] 0.40-1.63), and the rate of CJD did not increase over time (5-year RR 0.92, 95% CI 0.73-1.16). In Colorado, human prion disease resulting from CWD exposure is rare or nonexistent. However, given uncertainties about the incubation period, exposure, and clinical presentation, the possibility that the CWD agent might cause human disease cannot be eliminated.

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