Chronic Wasting Disease: Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene

Coding Region

Paula Stewart1, Lauren Campbell1, Susan Skogtvedt2, Karen A. Griffin3, Jon M. Arnemo4, Morten Tryland5,6, Simon Girling7, Michael W. Miller3, Michael A. Tranulis2, Wilfred Goldmann1* 1 Neurobiology Division, The Roslin Institute & R(D)SVS, University of Edinburgh, Roslin, United Kingdom, 2 Norwegian School of Veterinary Science, Dept. Basic Sciences & Aquatic Medicine, Oslo, Norway, 3 Wildlife Research Center, Colorado Division of Parks and Wildlife, Fort Collins, Colorado, United States of America, 4 Department of Forestry & Wildlife Management, Faculty of Applied Ecology and Agricultural Sciences, Hedmark University College, Campus Evenstad, Elverum, Norway & Department of Wildlife, Fish and Environmental Studies, Faculty of Forest Sciences, Swedish University of Agricultural Sciences, Umea°, Sweden, 5 Norwegian School of Veterinary Science, Section of Arctic Veterinary Medicine, Tromsø, Norway, 6 GenØk - Centre for Biosafety, Tromsø, Norway, 7 Royal Zoological Society of Scotland, Edinburgh Zoo, Edinburgh,

United Kingdom

Abstract

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrPC) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrPC protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.

Citation: Stewart P, Campbell L, Skogtvedt S, Griffin KA, Arnemo JM, et al. (2012) Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region. PLoS ONE 7(12): e50623. doi:10.1371/journal.pone.0050623 Editor: Mark D. Zabel, Colorado State University, College of Veterinary Medicine and Biomedical Sciences, United States of America Received August 29, 2012; Accepted October 23, 2012; Published December 7, 2012 Copyright: 2012 Stewart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: PS, LC and WG were supported by Roslin Institute Strategic Grant funding from the Biotechnology and Biological Sciences Research Council, United Kingdom. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wilfred.goldmann@roslin.ed.ac.uk

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Discussion

Prion diseases have not been observed in wild populations of carnivores, but some species of this order can become infected with prions and will develop clinical disease, prominent examples are FSE in cats and TME in mink. There is also no doubt that some carnivores are naturally exposed through their prey or carrion, such as to CWD infected deer and elk in the USA and Canada. However, there has been little research on the genetic susceptibility of carnivores to prion diseases and whether natural genetic variation in the prion gene is likely to influence transmission and susceptibility. In this study we have analysed the PRNP open reading frame sequences from a large number of species including FSE cases and evaluated PRNP sequence variability in wild populations.

Presence of PrPC is essential for the development of TSEs and dog, cat and mink do express detectable levels of PrPC [47,48]. Our data confirm this and reveal that PrPC in these species, including in bear and seal, is processed normally by proteolytic cleavage into fragments, which are equivalent to those seen in prion disease susceptible species such as sheep and man. Not only are these fragments produced but they also appear at ratios similar to other mammals; it remains to be shown whether different PrPC fragment ratios between canine and feline species underpin the different susceptibilities.

The lack of PrP variants associated with FSE-infected cats and cheetahs indicated that these animals were not at higher genetic risk than other individuals of their species. It can therefore be inferred –possibly with the inclusion of the lions- that FSE risk in the feline species was not controlled by highly susceptible PrP variants. The opposite, that most cats are genetically resistant through the presence of a common, ‘‘protective’’ PrP allele that is different to the susceptible ‘‘wildtype’’ allele can also be rejected, as our analysis of 68 UK cats and 118 cats in total should have revealed with 99.9% probability any alleles with frequencies equal to or higher than 5% and 3%, respectively [46]; the 4-repeat allele is not frequent enough to fulfil this role although we cannot exclude that it may have some protective effect.

The small number of FSE cases may be a consequence of low exposure, low natural transmission probability and possibly a large degree of underreporting of cases from a population in the millions, but it is also possible that non-PRNP genes with protective phenotype are particularly advantageous in felines or that fixed amino acid changes in the feline wildtype PrP sequence make cats partially resistant to prions. Although it could be argued that the considerable number of FSE cases in the relative small number of large cats in zoo collections does not support this hypothesis, they may have been exposed to a larger dose of prion infectivity than their domestic counterparts. The addition of alanine into the Nterminal repeats represents a feline PrPC specific change. It is difficult to predict its contribution to susceptibility. Although the repeats are not essential for replication of the agent or the PrPC to PrPSc conversion [49], they can modulate disease phenotypes such as PrPSc accumulation and incubation periods [49,50].

Cats may show average susceptibility or even partial resistance, in which case one has to ask whether the failure to find canine BSE cases has been a reflection of exposure, surveillance, diagnostics or genetics. There appears to be a strong case for a genetic/ biochemical explanation because in vitro amplification experiments with canid PrP showed reduced conversion ability [44,45]. By expanding the PRNP sequence analysis to large number of felids and canids we have shown that differences are consistently present in two regions of PrP: in codons 102f/99c, 110f/107c, 119f/116c and in codons 166f/163c, 184f/181c and 192f/189c. The substituted amino acids (Ser99c, Asn107c and Val116c) in canids have all been found in other species that are susceptible to prions and are unlikely to explain the different BSE susceptibility between felids and canids. The three C-terminal substitutions may be more illuminating as they show specificity for the respective Families. The variation between Arg192f and Lys189c (with Lys the most commonly found amino acid in the equivalent position in other species) can be regarded as conservative and may lead to minimal structural change in the protein, nevertheless it is worth exploring whether PrP with Arg192 changes susceptibility of Felidae to prion disease. Both Asp163c and Arg181c appear to be conserved ‘‘signature’’ amino acids for all Canidae as they were present in five genera. These major substitutions of Asn with Asp or Glu and His with Arg in highly conserved positions [30] are close to a b– strand (Asp163) and loop (Arg181) structures that appear to be critical in PrPC to PrPSc conversion (Figure S1) [51]. All three substitutions (Asp, Glu and Arg) result in changes of the charge distribution of PrPC at cellular pH and may lead to different interand intramolecular interactions [52]; when Asn was replaced with Asp in the equivalent codon of mouse PrPC (codon 158) in transgenic mice they were unable to produce PrPSc after prion challenge [53].

A thorough analysis of these and similar transgenic models in combination with in vitro studies of ‘‘signature substitutions’’ such as canine codon 163 or Arg192f will eventually reveal the underlying molecular mechanisms of prion replication. In contrast to the taxonomical relationship between Canidae and Mustelidae, the European wolverine and its close relative the Pine marten share the 163Asp substitution with canine PrP, whereas the omnivorous European badger, also a close relative to the wolverine does not. Hence, wolverines as predators of smaller ruminants and consumers of carrion may have similar genetic susceptibility as canids. The fixation of this asparagine in the wolverine and pine marten sequence is puzzling. How likely is it that 163Asp became fixed due to selection for PrPC rather than by random genetic drift? Studies in sheep investigating the influence of preclinical prion diseases on fitness and reproductive are not conclusive in this regard [54]. Genetic drift is however supported by the fact that 163Asp is seen in PrPC from an unrelated third species, the insectivorous little brown bat (Myotis lucifugus, Access. No. BN000992).

All animals from wild populations for which we analysed a larger number of samples (bear, wolf, mountain lion, seal) showed a very limited variation of their PrPC sequences. No amino acid polymorphisms were found in PRNP of mountain lion and black bear, only one dimorphism each was seen in brown bears, Gray wolves, coyotes, wolverines and three dimorphisms were found in Hooded Seals. This is similar to wild populations such as red deer (Cervus elephus) and roe deer (Capreolus capreolus) [55], wapiti (Cervus elaphus nelsonii) (Goldmann, unpublished observation) and fallow deer (Dama dama) [56]. The sample sizes of any of our investigated groups were small so that allele frequencies are only estimates, but they should reveal alleles with lowest frequencies of 3–10% with 99% probability. In contrast to the large number of PrPC variants maintained in livestock species such as sheep, sampling of companion animals such as cats and dogs did not appear to indicate an enhanced number of PrPC variants compared to the wild carnivores. Whereas PRNP in sheep may be under balancing selection [7] it appears more likely that it is under purifying selection in free ranging populations but this needs to be formally proven. We observed a slightly larger number of PrP variants in the family Canidae than in the Felidae and this appeared to hold true for wild and domestic animals. It has been shown for different prion diseases that PRNP gene heterozygosity can reduce susceptibility and lengthen the incubation period, consequently it appears that as individuals and at the population level canine species like wolf and coyote may have a double genetic advantage (number of PrP variants and the preservation of variants with low conversion efficiency) over bear and puma with regard to overall CWD susceptibility.

This study has shown that FSE cases were not associated with PRNP genetics and that wild populations of carnivores have very little genetic variation in the PRNP gene which could result in unrestricted susceptibility to TSEs. Genetic uniformity of PRNP in American black bear and mountain lion could result in homogeneous susceptibility to a compatible prion strain within these species - should such a strain emerge –.

Materials and Methods Ethics Statement

snip...see full text ;

http://www.plosone.org/article/fetchObjectAttachment.action;jsessionid=CEBB234EAA5A19106DA2610C9DC2BDC3?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0050623&representation=PDF

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0050623

Subject: CWD TSE PRION, AND SCRAPIE ?

*** Spraker suggested an interesting explanation for the occurrence of CWD. The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr. Bob Davis. At or abut that time, allegedly, some scrapie work was conducted at this site. When deer were introduced to the pens they occupied ground that had previously been occupied by sheep.

http://collections.europarchive.org/tna/20080102193705/http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf

White-tailed Deer are Susceptible to Scrapie by Natural Route of Infection

Jodi D. Smith, Justin J. Greenlee, and Robert A. Kunkle; Virus and Prion Research Unit, National Animal Disease Center, USDA-ARS

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. Previous experiments demonstrated that white-tailed deer are susceptible to sheep-derived scrapie by intracranial inoculation. The purpose of this study was to determine susceptibility of white-tailed deer to scrapie after a natural route of exposure. Deer (n=5) were inoculated by concurrent oral (30 ml) and intranasal (1 ml) instillation of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. Non-inoculated deer were maintained as negative controls. All deer were observed daily for clinical signs. Deer were euthanized and necropsied when neurologic disease was evident, and tissues were examined for abnormal prion protein (PrPSc) by immunohistochemistry (IHC) and western blot (WB). One animal was euthanized 15 months post-inoculation (MPI) due to an injury. At that time, examination of obex and lymphoid tissues by IHC was positive, but WB of obex and colliculus were negative. Remaining deer developed clinical signs of wasting and mental depression and were necropsied from 28 to 33 MPI. Tissues from these deer were positive for scrapie by IHC and WB. Tissues with PrPSc immunoreactivity included brain, tonsil, retropharyngeal and mesenteric lymph nodes, hemal node, Peyer’s patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by potential natural routes of inoculation. In-depth analysis of tissues will be done to determine similarities between scrapie in deer after intracranial and oral/intranasal inoculation and chronic wasting disease resulting from similar routes of inoculation.

see full text ;

http://www.usaha.org/Portals/6/Reports/2010/report-cwal-2010.pdf

PO-039:

A comparison of scrapie and chronic wasting disease in white-tailed deer

Justin Greenlee, Jodi Smith, Eric Nicholson US Dept. Agriculture; Agricultural Research Service, National Animal Disease Center; Ames, IA USA

http://www.landesbioscience.com/journals/prion/03-Prion6-2-Transmission-and-strains.pdf

White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation snip... It is unlikely that CWD will be eradicated from free-ranging cervids, and the disease is likely to continue to spread geographically [10]. However, the potential that white-tailed deer may be susceptible to sheep scrapie by a natural route presents an additional confounding factor to halting the spread of CWD. This leads to the additional speculations that 1) infected deer could serve as a reservoir to infect sheep with scrapie offering challenges to scrapie eradication efforts and 2) CWD spread need not remain geographically confined to current endemic areas, but could occur anywhere that sheep with scrapie and susceptible cervids cohabitate. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation with a high attack rate and that the disease that results has similarities to CWD. These experiments will be repeated with a more natural route of inoculation to determine the likelihood of the potential transmission of sheep scrapie to white-tailed deer. If scrapie were to occur in white-tailed deer, results of this study indicate that it would be detected as a TSE, but may be difficult to differentiate from CWD without in-depth biochemical analysis.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199251/?tool=pubmed

http://chronic-wasting-disease.blogspot.com/2011/10/white-tailed-deer-are-susceptible-to.html

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The familial mutations, Gajdusek proposed, lowered the barrier to such accidental conversion. "Thus," he wrote in 1996, "with these mutations, this ordinarily rare event becomes a ... dominant inherited trait." But Weissmann's qualification still remained to be refuted: the mutations might simply allow easier entry to a lurking virus. ...page 202 Deadly Feast

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something to think about for sure.

but i interpret this as (1st not the gold standard, just my opinion;-), as because of certain gene mutations, one or a family, would be more susceptible to the many different strains of TSE, and the many different proven routes and sources, (which will cause different symptoms, different incubation periods from onset of clinical symptoms to death, different parts of the brain infected, etc.). in other words, it's NOT the gene mutation that CAUSES the disease, but the fact that it makes you more SUSCEPTIBLE, to the TSEs from the surrounding environment, and PLUS accumulation, i think this plays a critical role. maybe there is a one dose scenario, but i think there is more of the 'accumulators' that go clinical, than the 'one dose'. and what is the threshold to sub-clinical to clinical ?

anyway, just pondering out loud here.

also, for anyone interested, there are some studies with links to follow here ;

http://sporadicffi.blogspot.com/

Thursday, September 27, 2012

Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease

http://chronic-wasting-disease.blogspot.com/2012/09/genetic-depletion-of-complement.html

Thursday, June 21, 2012

Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism

http://bse-atypical.blogspot.com/2012/06/clinical-and-pathologic-features-of-h.html

Friday, November 23, 2012

sporadic Creutzfeldt-Jakob Disease update As at 5th November 2012 UK, USA, AND CANADA

http://creutzfeldt-jakob-disease.blogspot.com/2012/11/sporadic-creutzfeldt-jakob-disease.html