molecular mechanisms of chronic wasting disease prion...

Molecular Mechanisms of Chronic WastingDisease Prion Propagation

Julie A. Moreno and Glenn C. Telling

Prion Research Center (PRC) and the Department of Microbiology, Immunology, and Pathology,Colorado State University, Fort Collins, Colorado 80525

Correspondence: [email protected]

Prion disease epidemics, which have been unpredictable recurrences, are of significantconcern for animal and human health. Examples include kuru, once the leading cause ofdeath among the Fore people in Papua New Guinea and caused by mortuary feasting; bovinespongiform encephalopathy (BSE) and its subsequent transmission to humans in the form ofvariant Creutzfeldt–Jakob disease (vCJD), and repeated examples of large-scale priondisease epidemics in animals caused by contaminated vaccines. The etiology of chronicwasting disease (CWD), a relatively new and burgeoning prion epidemic in deer, elk, andmoose (members of the cervid family), is more enigmatic. The disease was first described incaptive and later in wild mule deer and subsequently in free-ranging as well as captive RockyMountain elk, white-tailed deer, and most recently moose. It is therefore the only recognizedprion disorder of both wild and captive animals. In addition to its expanding range of hosts,CWD continues to spread to new geographical areas, including recent cases in Norway. Theunparalleled efficiency of the contagious transmission of the disease combined with highdensities of deer in certain areas of North America complicates strategies for controllingCWD and raises concerns about its potential spread to new species. Because there is a highprevalence of CWD in deer and elk, which are commonly hunted and consumed by humans,the possibilityof zoonotic transmission is particularly concerning. Here, we review the currentstatus of naturally occurring CWD and describe advances in our understanding of its molec-ular pathogenesis, as shown by studies of CWD prions in novel in vivo and in vitro systems.

EPIDEMIOLOGY AND HOST RANGE

Chronic wasting disease (CWD) was firstidentified in the late 1960s as a fatal wasting

syndrome of mule deer (Odocoileus hemionushemionus) and black-tailed deer (Odocoileushemionus columbianus) held in captivity in sev-eral wildlife facilities in northern Colorado. Bythe late 1970s, CWD was histopathologicallyrecognized as a prion disease by the late

Elizabeth Williams (Williams and Young 1980,1992). A retrospective study also revealed CWDinfection of mule and black-tailed deer that re-sided at the Toronto Zoo between 1973 and2003 (Dube et al. 2006). The disease was alsoidentified in mule deer in a research facility inWyoming and in captive wapiti, or RockyMountain elk (Cervus elaphus nelsoni), in boththe Colorado and Wyoming wildlife facilities.Thereafter, CWD was described in free-ranging

Editor: Stanley B. Prusiner

Additional Perspectives on Prion Diseases available at www.perspectivesinmedicine.org

Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a024448

1

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org

on May 23, 2020 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from

mailto:[email protected]



http://www.perspectivesinmedicine.org



http://perspectivesinmedicine.cshlp.org/

mule deer and elk in southeastern Wyomingand northeastern Colorado (Williams andYoung 1980, 1982, 1992) and was described inwhite-tailed deer in Nebraska and South Dako-ta in 2001 (Williams 2005). Surveillance andmodeling studies indicated that CWD occurredendemically among free-ranging deer and elk ina contiguous area encompassing northeasternColorado, southeastern Wyoming, and westernNebraska, and that CWD was most likelypresent in free-ranging cervids in this “endemicregion” several decades before its detection(Miller et al. 2000). Long thought to be limitedto this endemic region, a new area was identifiedin Saskatchewan, Canada (Williams and Miller2002), in 1996, and CWD also emerged in free-ranging populations of white-tailed deer (Odo-coileus virginianus) in southern Wisconsin (Jolyet al. 2003) in 2002. Most recently, CWD waspresent in a wild moose (Alces alces shirasi)killed by a hunter in Colorado (Baeten et al.2007) and in captive moose in Wyoming(Kreeger et al. 2006). The disease was subse-quently found in additional free-ranging moosein Colorado and Wyoming and most recently inAlberta, Canada.

The spread of CWD in North America ap-pears to be irrevocable. At the time of thisreview, CWD has been found in both wild andfarm-raised cervids from 22 North Americanstates, two Canadian provinces, and in Norway.Identification of CWD-infected animals in ar-eas previously thought to be free of infectionmay be partly related to increased surveillanceand spread of the disease by natural migrationand by humans who transport infected cervidsto other locations. These factors almost certain-ly play a role in the emergence of CWD in newareas. Moreover, this appears to be how out-breaks of the disease occurred in elk in theRepublic of Korea in 2001, 2004, and 2005—as a result of the country’s importation of sub-clinically infected animals (Sohn et al. 2002;Kim et al. 2005). In 2010, additional CWD caseswere observed in red deer (Cervus elaphus), sikadeer (Cervus nippon), and crossbred sika andred deer (Lee et al. 2013).

In April 2016, CWD was detected for thefirst time in Euorpe and for the first time

in free-ranging reindeer (Rangifer tarandustarandus) (Benestad et al. 2016). Western blotanalysis of brain material from this Norwegiananimal, which was from the Selbu region of Sør-Trøndelag county (Norway), showed that PrPSc

(the pathogenic form of the normal cellularprotein) glycosylation and molecular weightprofiles were similar to PrPSc from North Amer-ican elk with CWD. It was also found that PrPSc

deposition patterns were concordant with thoseof PrPSc in diseased reindeer orally inoculatedwith North American CWD (Mitchell et al.2012). In subsequent months, CWD was diag-nosed in a second reindeer from the Selburegion, as well as in two moose from the Nord-fjella region in southern Norway, which is sep-arated from Sør-Trøndelag by several hundredkilometers (S Benestad, pers. comm.). The originof these new European cases is currently unclear.

PATHOGENESIS

Clinical signs of infected deer and elk can besubtle, which makes diagnosis difficult, and in-clude weight loss, behavioral alterations, appar-ent ruminal atony, and salivary defluxion inlater stages of the disease. Clinical features in-clude gradual loss of body condition, resultingin emaciation (hence the term “wasting dis-ease”), and behavioral changes that include gen-eralized depression and loss of fear of humans(Williams 2003). At later stages, infected ani-mals may display polydipsia and polyuria, sia-lorrhea, and generalized incoordination. Theclinical course in captive animals is slowly pro-gressive, and after diagnosis, most animals sur-vive for a few weeks up to 3 to 4 mo.

Within a short time after an animal ingestsmaterial contaminated with CWD prions, thepathogenic prions migrate to lymphoid centersassociated with the alimentary tract, includingthe tonsil and retropharyngeal lymph nodes(Sigurdson et al. 2002). Following neuroinva-sion, CWD prions travel throughout the centralnervous system (CNS) via the ascending fibersof the autonomic nervous system, culminatingin prion deposits and spongiform degenerationin the dorsal motor nucleus of the vagus nervein the obex region of the medulla oblongata.

J.A. Moreno and G.C. Telling

2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a024448

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Ultimately, prions replicate throughout theCNS. Like other prion diseases, postmortempathognomonic lesions are confined to theCNS and consist of intraneuronal vacuolation,neuropil spongiosis, astrocytic hypertrophy,and hyperplasia (Williams and Young 1993).CWD is characterized by extensive depositionof PrPSc prions in the CNS and lymphoid tissue,the latter being detectable in the early stages ofthe disease (Sigurdson et al. 1999; Fox et al.2006). The pathogenesis of CWD seems tovary between deer and elk, with fewer depositsseen in the lymphoid tissue of elk comparedwith deer (Race et al. 2007). Florid amyloidplaques also feature in the neuropathology ofdiseased deer (Liberski et al. 2001). Othertissues in which PrPSc or infectivity has beendetected in deer and elk include the pancreas(Sigurdson et al. 2001; Fox et al. 2006), adrenalgland (Sigurdson et al. 2001; Fox et al. 2006),and cardiac muscle (Jewell et al. 2006). CWDprions have also been detected in saliva andblood by bioassay (Mathiason et al. 2006) andin urine by protein-misfolding cyclic amplifica-tion (PMCA) (Haley et al. 2009a) and bioassay(Haley et al. 2009b), which suggests that thesebody fluids play a role in the transmission anddissemination of the disease. The fecal materialof subclinical deer also harbors infectivity (Ha-ley et al. 2009b; Tamguney et al. 2009). Please seeHaley and Hoover (2015), who have recentlyreviewed pathogenesis in the natural host, foran exhaustive description of the clinical andpathological features of the disease.

NATURAL TRANSMISSION

The maximum incubation period of CWD innaturally infected animals is unknown, butmost natural cases occur in animals 3 to 7 yrold, with the majority of animals probably de-veloping the disease within 3 yr of infection(Miller et al. 1998). Although CWD has beenshown to be experimentally transmissible afterintracerebral inoculation of mule deer withincubation periods up to 2 yr (Williams andYoung 1992), limited transmission studieshave indicated that CWD developed �25%more rapidly in orally challenged elk than deer

(16 mo for mule deer and 12 mo for elk) (Wil-liams 2003). Levels of PrPSc were found to behigher in the tonsil and retropharyngeal lymphnodes of CWD-infected deer than elk (Raceet al. 2007). Because this finding correlateswith the natural prevalence of CWD in thesespecies, Race et al. speculated that CWD-infect-ed deer may be more likely than elk to transmitthe disease.

In the wild, the highly efficient transmissionof CWD appears unparalleled among prion dis-eases (Williams and Young 1980; Miller et al.2000; Miller and Williams 2003). The remark-ably contagious nature of CWD has been doc-umented in a captive mule deer population,wherein 90% of the mule deer present in thepopulation for .2 yr ultimately developed thedisease (Williams and Young 1980). Althoughthe natural route of transmission is not preciselyknown, lateral transmission (Williams and Mil-ler 2002) by ingesting forage or water contam-inated by secretions, excretions, or other sources(for example, CWD-infected carcasses) (Milleret al. 2004) has long been thought to be the mostplausible natural route. The presence of CWDprions in saliva, blood, urine, and feces (Haleyet al. 2009a,b, 2011; Mathiason et al. 2006; Tam-guney et al. 2009) is consistent with this mech-anism of contagious lateral transmission. Usingtransgenic (Tg) mice bioassays, CWD prionswere detected in elk antler velvet, and the annualshedding of this material increases the possibil-ity that it may also play a role in transmitting thedisease (Angers et al. 2009). Recently developedhighly sensitive methods for CWD detectionbased on a standardized real-time quaking-in-duced conversion (RT-QuIC) assay are likely togreatly facilitate epidemiological studies (Haleyet al. 2014; Henderson et al. 2015).

Pertinent to the issue of disease transmis-sion is the seemingly indefinite persistence ofCWD in the environment (Miller et al. 2004;Seidel et al. 2007), a feature that is linked tothe unusual and extreme resistance of prionsto degradation. Coupled with this characteris-tic, prions bind avidly to soil particles, particu-larly montmorillonite clay and quartz (Smithet al. 2011), and remain infectious after oralconsumption (Johnson et al. 2006a, 2007;

Molecular Mechanisms of CWD Prion Propagation

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a024448 3

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Saunders et al. 2012). This binding requires theflexible N terminus of PrPSc and cleavage withproteinase K effectively releases prions from soilparticles (Saunders et al. 2011). Of note, thereappears to be a close correlation between thesesoil types and CWD prevalence (Saunders et al.2012).

Shedding of CWD prions during infectionraised the possibility that contamination ofplant materials may feature in disease transmis-sion. To address this, Pritzkow and colleagues(2015) analyzed the binding and retention ofPrPSc to plants. Their study found that evensmall quantities of prions in diluted brainhomogenate or in urine and feces bind to wheatgrass roots and leaves for several weeks, and thatanimals are efficiently infected by ingesting pri-on-contaminated plants. These features applyto prions from diverse origins, including fromCWD. Moreover, root uptake of prions fromcontaminated soil is followed by their transportto the stem and leaves.

IMPACT AND MANAGEMENT OF CWD

Although most U.S. states and Canadian prov-inces have introduced CWD surveillance pro-grams, the extent of these programs is variable,ranging from targeted surveillance in some statesto mandatory testing of all animals suspected ofdying of CWD in others. Complicating the issue,diagnosis can only be unequivocally made fol-lowing postmortem analysis of CNS materials,and current evaluations almost certainly under-estimate the true prevalence of the disease. Al-though testing for CWD in other countries hasbeen minimal, limited active surveillance has, todate, not detected CWD in European countriesother than Norway or Japan (Roels et al. 2004;Kataoka et al. 2005; Schettler et al. 2006).

The prevalence of CWD can be as highas 30% in some areas of Colorado (Williams2005), and wildlife management efforts to con-tain or eradicate CWD in the state have provenunsuccessful (Conner et al. 2007). Based onhunter-harvested animal surveillance, the prev-alence of CWD in the endemic area from 1996 to1999 was estimated at �5% in mule deer, �2%in white-tailed deer, and ,1% in elk (Spraker

et al. 1997). Several studies have attempted toaddress the impact of CWD on cervid popula-tions. Experimental studies show that CWDlowers deer survival and can depress populationgrowth rates when prevalence becomes suffi-ciently high (Miller et al. 2008; Dulberger et al.2010). Modeling approaches have provided con-flicting predictions of the consequences of CWDon infected populations (Wasserberg et al. 2009;Almberg et al. 2011; Geremia et al. 2015).

CWD has both a significant economic im-pact and influences wildlife conservation. TheU.S. Fish and Wildlife Service estimated that atotal of $33.7 billion was spent on hunting itemsin 2011, and an estimated 11.6 million hunterspursued big game such as deer and elk (U.S.Census Bureau 2011). Within the first monthof diagnosing CWD in free-ranging deer in2002, Wisconsin’s wildlife management agencyspent �$250,000 in control and public informa-tion efforts and subsequently upward of $2.5millionayear foradditional CWDcontrolefforts(Telling 2013). Saskatchewan has spent �$30million to eradicate the disease within infectedcommercially operated game farms (OklahomaDepartment of Wildlife Conservation 2002).

EXPERIMENTAL TRANSMISSION OF CWD

Other cervid species are susceptible to CWDfollowing experimental challenge. These in-clude European red deer (Cervus elaphus ela-phus) (Martin et al. 2009) and muntjac deer(Muntiacus reevesi) (Nalls et al. 2013). Braintissue from CWD-infected white-tailed deerand elk produced disease in only four of 13intracerebrally inoculated fallow deer (Damadama) (Hamir et al. 2008), and the same speciesappeared resistant when cohoused in paddockswith CWD-infected mule deer (Rhyan et al.2011), suggesting relative resistance of this cer-vid species to CWD.

Whether the natural host range of CWDextends beyond the family Cervidae is currentlyunclear. However, the remarkably high rate ofCWD prion transmission brings into questionthe risk posed to livestock of developing a novelCWD-related prion disease via shared grazingof CWD-contaminated rangeland. This issue



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



has been addressed by transmitting CWD to cat-tle (Hamir et al. 2001, 2005, 2006a, 2007a) andsheep (Hamir et al. 2006b). In the case of cattle,PrPSc was detected in �40% of intracerebrallyinoculated animals. Low rates of disease alsooccurred in intracerebrally inoculated sheep.Host genotype appeared to affect the efficiencyof transmission. Tg mice expressing ovine orbovine prion protein (PrP) have also been chal-lenged with CWD, thus far with negative out-comes (Tamguney et al. 2006). Davenport et al.(2015) used the RT-QuIC assay to comparethe conversion properties of CWD and bovinespongiform encephalopathy (BSE) prions, aswell as feline-adapted versions of these prions.CWD, BSE, and feline-adapted CWD prionsmost effectively seeded cervid, bovine, andfeline bacterially expressed recombinant PrP(rPrP), respectively, whereas feline spongiformencephalopathy (FSE) prions more efficientlyconverted bovine than feline rPrP. To determinethe potential of these prions to convert humanPrP, human rPrP was used as a substrate inRT-QuIC. Remarkably, CWD, feline-adaptedCWD, BSE, and FSE prions all converted humanrPrP, although not as efficiently as sporadicCreutzfeldt–Jakob disease (CJD) prions.

Experimental transmission to other specieshas shown mixed results. Studies by the late Dr.Richard Marsh in the mid-1980s at the Univer-sity of Wisconsin showed that the CWD agenttransmitted poorly to Syrian golden hamsters,ferrets, and mink (Bartz et al. 1998; Marsh et al.2005; Sigurdson et al. 2008). Other susceptiblespecies include several species of voles, white-footed mice, deer mice, cats, raccoons, andsquirrel monkeys (Hamir et al. 2003, 2007b;Race et al. 2009a, 2014; Heisey et al. 2010; DiBari et al. 2013; Mathiason et al. 2013; Seeliget al. 2015). Although non-Tg mice have beenreported to be resistant to CWD infection(Browning et al. 2004), limited infection ofthe VM/Dk inbred strain of mice with elkCWD prions has been reported (Lee et al. 2013).

TRANSGENIC MOUSE MODELS OF CWD

Although CWD is transmissible after intracere-bral inoculation of mule deer with incubation

periods up to 2 yr (Williams and Young 1992),the expense of housing cervids under prion-freeconditions for long periods and the highly com-municable nature of CWD present significantchallenges for using deer as experimental hosts(Mathiason et al. 2006). As noted above, CWDtransmissions to other species have yielded mixedresults, but are generally inefficient, albeit tovarying extents, an effect referred to as the speciesbarrier to prion transmission (Pattison 1965).

Seminal studies in Tg mice with sheep scra-pie prions experimentally adapted to mice orSyrian hamsters showed that optimal diseaseprogression requires that the primary structuresof the pathogenic form of the prion protein(PrP), referred to as PrPSc, and the normal cel-lular form, referred to as PrPC, are related (Pru-siner et al. 1990; Scott et al. 1993). These studiespaved the way for the development of Tg modelsin which to study human prions (Telling et al.1995) and subsequently other naturally occur-ring mammalian prions (for review, see Telling2011). Studies in Tg mice also underscored thatthe tertiary structure of PrPSc enciphers straininformation (Bessen and Marsh 1994; Telling etal. 1996). Additionally, Tg mice have been usedto model interspecies transmission (Vickery etal. 2014), which is generally inefficient and usu-ally characterized by low attack rates with longand variable incubation times upon primarytransmission. Relatively facile transmissionupon serial passage reflects adaptation in thenewly infected species (Telling 2011). The initialbarrier to propagation is thought to result fromprimary structural incompatibilities betweendonor PrPSc and recipient PrPC, resulting in in-efficient PrPC conversion. Ultimately, nascentPrPSc composed of the new host primary struc-ture readily converts PrPC, and efficient prionpropagation ensues in the recipient species.

Using this rationale, several Tg mouse mod-els expressing either elk or deer PrP have beenproduced in which the species barrier to CWDhas been eliminated. Prototype Tg mice express-ing deer PrP, designated Tg(CerPrP)1536þ/ –

(Browning et al. 2004) and referred to here asTg(DeerPrP), recapitulated the cardinal neuro-pathological, clinical, and biochemical featuresof CWD, an observation subsequently con-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



firmed in comparable Tg mouse modelsexpressing deer or elk PrP (Kong et al. 2005;LaFauci et al. 2006; Tamguney et al. 2006;Meade-White et al. 2007; Angers et al. 2009).The generation of CWD-susceptible Tg mice, inconcert with the development of PMCA-basedapproaches for amplifying CWD infectivity us-ing PrPC expressed in the CNS of those mice(Green et al. 2008a; Meyerett et al. 2008) hasalso provided crucial information about the bi-ology of CWD and cervid prions. Amplificationin vitro was shown to maintain CWD prion-strain properties and provided a means of gener-ating novel cervid prion strains (Kurt et al. 2007,2009; Green et al. 2008a; Meyerett et al. 2008).

Studies in Tg mice and in vitro amplifica-tion approaches have also facilitated our under-standing of the mechanism of CWD transmis-sion among deer and elk (Mathiason et al. 2006;Haley et al. 2009a,b; Tamguney et al. 2009).Transmission studies in Tg(DeerPrP) and sim-ilar lines of Tg mice showed that CWD prionswere present in urine, feces, and saliva (Tamgu-ney et al. 2006; Haley et al. 2009a), and thesefindings were substantiated by in vitro amplifi-cation techniques (Haley et al. 2009a; Pulfordet al. 2012; Henderson et al. 2013).

Tg approaches in mice have been essential toassessing the potential risk of human exposureto CWD prions (Angers et al. 2006, 2009; Raceet al. 2009b). For the first time, the availability ofCWD-susceptible Tg mouse models has alsoprovided a means of quantifying CWD infectiv-ity by end-point titration (Angers et al. 2009).

GENERAL INSIGHTS INTO THE MECHANISMOF PRION PROPAGATION FROM CWD-SUSCEPTIBLE TRANSGENIC MOUSE MODELS

Effects of Polymorphisms in the UnstructuredN Terminus of PrP

As shown in other species in which prion dis-eases occur naturally, susceptibility to CWD ishighly dependent on polymorphic variation indeer and elk PRNP. Polymorphisms at codons95 (glutamine [Q] or histidine [H]) (Johnsonet al. 2003), 96 (glycine [G] or serine [S]) (Ray-mond et al. 2000; Johnson et al. 2003), and 116

(alanine [A] or G) (Heaton et al. 2003) in white-tailed deer have been reported. Although allmajor genotypes were found in deer withCWD, the Q96, G96, A116 allele (QGA) wasmore frequently found in CWD-affected deerthan the QSA allele (Johnson et al. 2003;O’Rourke et al. 2004), suggesting a protectiveeffect of the counterpart polymorphisms. Theelk PRNP coding sequence is polymorphicat codon 132, encoding either methionine (M)or leucine (L) (Schatzl et al. 1997; O’Rourkeet al. 1999). This position is equivalent to hu-man PRNP codon 129. Studies of free-rangingand captive elk with CWD (O’Rourke et al.1999), as well as oral transmission experiments(Hamir et al. 2006c; O’Rourke et al. 2007), in-dicate that the L132 allele protects against CWD.

Tg mouse modeling provides a means ofassessing the role of these cervid PrP gene poly-morphisms on CWD pathogenesis. In recentwork combining studies in Tg mice, the naturalhost, cell-free prion amplification, and molec-ular modeling approaches, we analyzed the ef-fects of deer polymorphic amino-acid varia-tions on CWD propagation and susceptibilityto prions from different species (Angers et al.2014). Reflecting the general authenticity ofthe Tg modeling approach, the properties ofCWD prions were faithfully maintained indeer following their passage through Tg miceexpressing cognate PrP. Moreover, the protec-tive influences of naturally occurring PrP poly-morphisms on CWD susceptibility were accu-rately reproduced in Tg mice or during cell freeamplification. The resistance to CWD of Tgmice expressing deer PrP with serine at residue96, referred to as Tg(DeerPrP-S96)7511 mice, isconsistent with previously generated tg60 miceexpressing serine at residue 96 (Meade-Whiteet al. 2007). In the studies of Angers andcolleagues, whereas substitutions at residues95 and 96 affected CWD propagation, theirprotective effects were overridden during repli-cation of sheep prions in Tg mice and, in thecase of residue 96, deer.

To more fully address the influence of the elk132 polymorphism, transmissibility of CWDprions was assessed in Tg mice expressing cervidPrPC with leucine or methionine at residue 132



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



(Green et al. 2008b). Although Tg mice express-ing leucine at residue 132 afforded partial resis-tance to CWD, Sheep Scrapie Brain Pool(SSBP)/1 sheep scrapie prions transmitted effi-ciently to these mice, suggesting that the elk 132polymorphism also controls prion susceptibil-ity at the level of prion-strain selection. Thecontrasting ability of CWD and SSBP/1 prionsto overcome the inhibitory effects of leucine atresidue 132 allele is reminiscent of studies de-scribing the effects of the human codon 129methionine (M)/valine (V) polymorphism onvCJD/BSE prion propagation in Tg mice ex-pressing human PrP, which concluded that hu-man PrP with valine at residue 129 severely re-stricts propagation of the BSE prion strain(Wadsworth et al. 2004). It therefore appearsthat amino-acid substitutions in the unstruc-tured region of PrP affect PrPC-to-PrPSc conver-sion in a strain-specific manner.

The susceptibility of Tg(DeerPrP-S96)7511mice, albeit with incomplete attack rates andlong incubation times, is at odds with previouswork showing complete resistance of tg60mice, which express the same deer PrP variant(Meade-White et al. 2007; Race et al. 2011). Thisapparent discrepancy is most likely related tothe low transgene expression in tg60 mice,which is reported to be 70% of the levels foundin deer. CWD occurs naturally in deer homozy-gous for the PrP-S96 allele (Keane et al. 2008),which is clearly inconsistent with this substitu-tion’s completely protective effect, suggestingthat Tg(DeerPrP-S96)7511 mice represent anaccurate Tg model in which to assess the effectsof the S96 substitution.

In accordance with the role of this region instrain selection, in subsequent studies, Tg miceexpressing wild-type deer PrP (tg33) or tg60were challenged with CWD prions from exper-imentally infected deer with varying polymor-phisms at residues 95 and 96 (Duque Velasquezet al. 2015). Passage of deer CWD prions intotg33 mice resulted in 100% attack rates, withCWD prions from deer expressing H95 or S96having significantly longer incubation periods.Remarkably, otherwise resistant tg60 mice(Meade-White et al. 2007; Race et al. 2011) de-veloped disease only when inoculated with pri-

ons from deer expressing H95/Q95 and H95/S96 PrP genotypes. Serial passage in tg60 miceresulted in propagation of a novel CWD strain,referred to as H95þ, whereas transmission totg33 mice produced two disease phenotypesconsistent with propagation of two strains.

Effects of Residues in a Discontinuous EpitopeFormed by Interactions between a b2–a2Loop and a-Helix 3

High-resolution structural studies showed thatthe loop region linking the secondb -sheet (b2)with thea 2-helix (a2) of cervid PrP is extreme-ly well defined compared with most otherspecies, increasing the possibility that this struc-tural characteristic correlates with the unusuallyfacile contagious transmission of CWD (Gos-sert et al. 2005). Tg mice expressing mouse PrPin which the b2–a2 loop was replaced by thecorresponding region from cervid species spon-taneously developed prion disease (Sigurdsonet al. 2009). Additional studies consistentlypoint to the importance of the b2–a2 loop inregulating transmission barriers, including thatof humans to CWD (Kurt et al. 2009, 2014,2015; Sigurdson et al. 2010).

Subsequent work suggested a more complexmechanism in which the b2–a2 loop partici-pates with the distal region of a-helix 3 to forma solvent-accessible contiguous epitope (Perezet al. 2010). These and later studies (Christenet al. 2009) ascribed greater importance to theplasticity of this discontinuous epitope. For ex-ample, substitution of aspartic acid (D) foundin horse, which contains a similarly structuredloop region, with serine (S) at residue 170 ofmouse (elk PrP numbering), increased notonly the structural order of the loop, but alsothe long-range interaction with tyrosine (Y) atresidue 228 in a-helix 3. Underscoring the im-portance of long-range b2–a2 loop/a-helix 3interactions, similar structural connections oc-cur between this residue, which is alanine (A) inTammar wallaby PrP, and residue 169 in theb2–a2 loop (Christen et al. 2009). Stabilizinglong-range interactions between the b2–a2loop and a-helix 3 also occur in rabbit PrP,a species generally regarded as resistant to



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



prion infection. X-ray crystallographic analysesshowed that the rabbit b2–a2 loop is clearlyordered and indicated that hydrophobic inter-actions between the side chains of valine (V) atresidue 169, and in this case Y221 (elk PrP num-bering) of a-helix 3, contributed to the stabilityof the b2–a2 loop/a helix 3 epitope (Khanet al. 2010).

In mule deer, polymorphism at codon 225encoding S or phenylalanine (F) influencesCWD susceptibility, with the 225F allele beingrelatively protective. The occurrence of CWDwas found to be 30-fold higher in deer homo-zygous for serine at position 225 (225SS) thanin heterozygous (225SF) animals; the frequencyof 225SF and 225FF genotypes in CWD-nega-tive deer was 9.3%, but only 0.3% in CWD-positive deer (Jewell et al. 2005). Recent studiescomparing CWD susceptibility in mule deerwith either of the residue 225 genotypes,225SS or 225FF, showed that 225FF mule deerhad differences in clinical disease presentationas well as subtler atypical traits (Wolfe et al.2014). Immediately adjacent to the protectivemule deer PrP polymorphism at 225, residue226 encodes the singular primary structural dif-ference between Rocky Mountain elk and deerPrP. Elk PrP contains glutamate (E) at this po-sition, and deer PrP contains glutamine (Q).

Recent findings show that residues 225 and226 play a critical role in PrPC-to-PrPSc conver-sion and strain propagation, but the effects aredistinct from those produced by the H95Q,G95S, and M132L polymorphisms (Angerset al. 2014). Structural analyses confirm thatresidues 225 and 226 are located in the distalregion of a-helix 3 that participates with theb2–a2 loop to form a solvent-accessible con-tiguous epitope (Perez et al. 2010). Consistentwith the role of this epitope in PrP conversion,these polymorphisms severely impact replica-tion of both SSBP/1 and, to variable degrees,CWD. In the case of Tg mice expressing deerPrP with phenylalanine at 225, referred to asTg(DeerPrP-F225), SSBP/1 incubation timeswere prolonged threefold, whereas inoculationwith CWD produced incomplete attack ratesand prolonged and variable incubation timesin the small numbers of mice that did develop

disease. In those Tg(DeerPrP-F225) mice thatdid succumb to CWD, PrPSc distribution pat-terns were altered compared with Tg(DeerPrP)mice.

To address the effects of the substitution ofglutamate for glutamine at residue 226, weassessed whether Tg mice expressing wild-typeelk or deer PrP differed in their responses toCWD. These studies showed that differences atresidue 226 also affected CWD replication, butto a lesser degree than the residue 225 polymor-phism, with disease onset prolonged by 20%–46% in CWD-inoculated Tg(DeerPrP) com-pared with Tg(ElkPrP) mice, and PrPSc distri-bution and neuropathology varying in each case(Angers et al. 2009). In contrast to Tg(DeerPrP)mice, which are susceptible to SSBP/1 (Greenet al. 2008b), Tg(ElkPrP) mice were completelyresistant (Angers et al. 2009), although the re-sistance of elk PrPC to propagation of SSBP/1was overcome following adaptation in deer orTg(DeerPrP) mice. Passage in Tg mice express-ing E226 or Q226 profoundly affected the abil-ity of SSBP/1 to reinfect Tg mice expressingsheep PrPC. These studies paralleled aspects ofour previously published work indicating thatamino-acid differences at residue 226 controlledthe manifestation of CWD quasispecies orclosely related strains (Angers et al. 2010). Thesefindings therefore collectively point to an im-portant role for residues 225 and 226 in PrPC-to-PrPSc conversion and the manifestation ofprion-strain properties. They substantiate theview that long-range interactions between theb2–a2 loop and a-helix 3 provide protectionagainst prion infection and suggest a likelymechanism to account for the protective effectsof the F225 polymorphism. Molecular dynam-ics analyses (Angers et al. 2014) showed that theS225F and E226Q substitutions in deer alter theorientations of D170 in the b2–a2 loop andY228 in a-helix 3. This structural change allowshydrogen bonding between the side chainsof these residues, which results in reduced plas-ticity of the b2–a2 loop/a-helix 3 epitopecompared with deer or elk PrP structures. Thissuggests that the increased stability of thistertiary structural epitope precludes PrPC-to-PrPSc conversion of deerPrP-F225.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



CELL CULTURE MODELS OF CWD

Therapeutic Approaches

Based on observations showing that rabbit kid-ney epithelial (RK13) cells engineered to expresssheep PrP were capable of propagating scrapieprions (Vilette et al. 2001), cloned RK13 cellsexpressing elk PrP were developed. A highlysusceptible clone producing PrPSc, referred toas Elk-21þ, was isolated in which CWD infec-tion was maintained for more than 100 passages(Bian et al. 2010). Inoculation of CWD-suscep-tible Tg(ElkPrP) mice with prions from Elk-21þ

cells resulted in disease transmission with clin-ical and neuropathological features identical toCWD.

This development provided a unique andconvenient means of exploring CWD therapeu-tics. Our first approach was to assess the efficacyof compounds with known effects on rodentprions. Consistent with its effect on mouse pri-on infectivity, sustained treatment of Elk-21þ

cells with dextran sulfate 500 (DS-500) resultedin PrPSc clearance, which did not reemerge inthe resulting Elk-212 cells after more than 40passages, and inoculation of susceptible Tgmice showed that Elk-212 cells were cured ofCWD prion infection (Bian et al. 2010). In starkcontrast to its well-documented inhibitory ef-fects on PrPSc accumulation in cells chronicallyinfected with experimentally adapted rodentprions (Doh-Ura et al. 2000; Korth et al. 2001;Barret et al. 2003; Ryou et al. 2003; Klingensteinet al. 2006; Fasano et al. 2008; Mays et al. 2012 ),quinacrine increased elk and deer PrPSc accu-mulation and raised prion titers in cells persis-tently infected with CWD prions (Bian et al.2014). These findings show that a compoundwith demonstrated, albeit transient, efficacyagainst a strain in one species acts to directlyenhance prion replication in a different speciesand produces prions with novel biologicalproperties. This means that the effects of “antip-rion” compounds are clearly species/strain de-pendent, in that drugs found to restrain prionsin one situation may improve replicative abilityin another. These findings force a reconsidera-tion of current strategies to screen antipriondrugs. In particular, it seems imperative that

cells with susceptibilities to cervid, ovine, bo-vine, and human prions be used to identifydrugs capable of treating prions affecting thosespecies.

Studies with the 2-aminothiazole referredto as IND24 showed that mice treated withthis compound and infected with mouse-adapted scrapie had longer survival times, butthat treatment led to the emergence of drug-resistant variants (Berry et al. 2013). IND24doubled the incubation times for Tg mice in-fected with CWD prions, and extensive analysesindicated that the CWD prions were not alteredby IND24 treatment. This is significant becausethese results reveal efficacy of a therapeutic on anaturally occurring prion, in a system replicat-ing such prions, and suggest that IND24 may bea viable candidate for treating CWD in naturalhosts (Berry et al. 2015).

With respect to therapeutic options forCWD, white-tailed deer orally vaccinated withattenuated Salmonella expressing PrP and orallychallenged with CWD had a significant prolon-gation of the incubation period, with one deerremaining asymptomatic by several sensitivecriteria (Goni et al. 2015).

Cell-Based CWD Quantification

Elk-212 cells were used to develop a novel cell-based assay for CWD prion quantification,analogous to the scrapie cell assay developedby Weissmann and colleagues (Klohn et al.2003) as a facile alternative to in vivo CWDprion quantification. This cell-based assay is re-ferred to as the cervid prion cell assay (CPCA).Detection and quantification of cervid prions,including naturally occurring CWD prions andexperimentally adapted cervid prion strains,was made possible using the CPCA (Bian et al.2010).

In the CPCA, CWD prion-susceptible Elk-212 cells in 96-well plates are exposed to serialdilutions of the prion-containing sample for4 d, grown to confluence, split at a ratio of 1:8,grown to confluence once more, and split sim-ilarly again. When the cells have reached con-fluence after the second split, 20,000 cells arefiltered onto membranes of Elispot plates, and



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



the proportion of cells containing protease-resistant CerPrPSc is identified by an enzyme-linked immunosorbent assay (ELISA) usingautomated counting equipment (Elispot). In-clusion of RK13 cells stably transfected withempty vector showed that positive spots detect-ed after three splits were the result of newlygenerated CerPrPSc. Although CerPrPSc purifi-cation as described for other CWD cell-culturesystems (Raymond et al. 2006) was not aprerequisite for sustained cellular infection, ex-pression of retroviral Gag facilitated prion sus-ceptibility, and cell cloning was also critical.

This cell-based approach also facilitatednovel means of assessing prion-strain proper-ties, specifically conformational stability analy-ses of PrPSc (Peretz et al. 2001). Apart fromlimited investigations in infected cell cultures(Ghaemmaghami et al. 2011), such analysesare generally conducted on PrPSc produced ininfected brains. This constraint relates, in largepart, to relatively low PrPSc levels in infected cellcultures and a paucity of sensitive cell-basedanalytical approaches. A cell-based conforma-tional stability assay (C-CSA) circumvents thesedrawbacks because it provides the advantage ofdirect in situ detection of PrPSc without isola-tion or purification and is a quantitative, sensi-tive, and accurate means of discerning the un-folding characteristics of PrPSc (Bian et al. 2014).

CWD STRAINS

Although our seminal studies in Tg mice(Browning et al. 2004) and subsequent work(LaFauci et al. 2006) raised the possibility ofCWD strain variation, the limited number ofisolates and the lack of detailed strain analysesin these studies means that this hypothesisremained speculative. Subsequent studies sup-ported the feasibility of using Tg(DeerPrP)mice for characterizing naturally occurringCWD strains, as well as novel cervid prions gen-erated by PMCA (Green et al. 2008a). To addresswhether different CWD strains occur in variousgeographical locations or in different cervidspecies, bioassays in Tg mice were used toanalyze CWD in a large collection of captiveand wild mule deer, white-tailed deer, and elk

from various geographical locations in NorthAmerica (Angers et al. 2010). These findingsprovided substantial evidence for two prevalentCWD prion strains, referred to as CWD1 andCWD2, with different clinical and neuropatho-logical properties. Remarkably, primary trans-mission of CWD prions from elk producedeither CWD1 or CWD2 profiles, whereas trans-mission of deer inocula favored the productionof mixed intra-study incubation times andCWD1 and CWD2 neuropathologies. Thesefindings indicate that elk may be infected witheither CWD1 or CWD2, whereas deer brainstend to harbor CWD1/CWD2 strain mixtures.

The different primary structures of deer andelk PrP at residue 226 provide a framework forunderstanding these differences in strain pro-files of deer and elk. Because of the role playedby residue 226, the description of a lysine (K)polymorphism at this position in deer (Johnsonet al. 2006b) and its possible role on strainstability may be significant. It is unknownwhether CWD1 and CWD2 interfere or act syn-ergistically or whether their coexistence con-tributes to the unparalleled efficiency of CWDtransmission. Interestingly, transmission resultsreported in previous studies suggest that cervidbrain inocula might be composed of strain mix-tures (Tamguney et al. 2006).

Additional studies support the existence ofmultiple CWD strains. CWD has also beentransmitted, albeit with varying efficiency, toTg mice expressing mouse PrP (Sigurdsonet al. 2006; Tamguney et al. 2006). In the Sigurd-son et al. study, a single mule deer isolateproduced disease in all inoculated Tga20 mice,which express mouse PrP at high levels. On suc-cessive passages, incubation times dropped to�160 d. In the second study, one elk isolatefrom a total of eight deer and elk CWD isolatesinduced disease in 75% of inoculated Tg4053mice, which also overexpress mouse PrP. Thedistribution of lesions in both studies appearedto resemble the CWD1 pattern. Low efficiencyCWD prion transmission was also recordedin hamsters and Tg mice expressing Syrianhamster PrP (Raymond et al. 2007). In thatstudy, during serial passage of mule deerCWD, fast- and slow-incubation-time strains



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



with different patterns of brain pathology andPrPSc deposition were also isolated. In otherstudies, serial passages of CWD from white-tailed deer into Tg mice expressing hamsterPrP, and then Syrian golden hamsters, produceda strain, referred to as “wasting” (WST), char-acterized by a prominent preclinical wastingdisease similar to cachexia, which the investiga-tors propose results from a prion-induced en-docrinopathy (Bessen et al. 2011). These sameinvestigators identified a second strain, definedas “cheeky” (CKY), derived from infection of Tgmice that express hamster PrP (Crowell et al.2015). The CKY strain had a shorter incubationperiod than WST, but after transmission tohamsters, the incubation period of CKY be-came �150 d longer than WST. In this case,PK digestion revealed strain-specific PrPSc sig-natures that were maintained in both hosts, butthe solubility and conformational stability ofPrPSc differed for the CWD strains in a host-dependent manner. In addition to supportingthe view that there are multiple CWD strains,these findings suggest the importance of host-specific pathways, independent of PrP, thatparticipate in the selection and propagation ofdistinct strains.

Studies in cell culture also support theexistence of CWD strains. Quinacrine alteredthe transmission properties of CWD prions,as well as the biochemical characteristics ofthe constitutive PrPSc (Bian et al. 2014). Despiteaccumulating significantly higher prion titers,quinacrine-treated (Q-CWD) prions fromElk-21þ cells produced prolonged incubationtimes in Tg(DeerPrP) and Tg(ElkPrP) micecompared with CWD prions in untreatedElk-21þ cells. Because the kinetics of diseaseonset in prion-infected animals is inversely re-lated to the titer of a given prion strain, thisunusual outcome is consistent with quinacrineaffecting the intrinsic properties of the CWDprion. In accordance with this notion, althoughthe deposition patterns of PrPSc in the brains ofdiseased Tg(DeerPrP) and Tg(ElkPrP) mice re-ceiving prions from Elk-21þ are concordantwith our previously published descriptions fol-lowing transmission of naturally occurring orPMCA-generated CWD prions (Green et al.

2008a), these distinctive patterns were not reca-pitulated in either line of Tg mice receivingQ-CWD prions. Although prion incubationtimes and neuronal targeting are the biologicalcriteria by which prion strains are classicallydefined, subsequent studies showed that strainproperties are enciphered within the conforma-tion of PrPSc (Telling et al. 1996; Green et al.2008a), and cervid prion incubation timespositively correlate with PrPSc conformationalstability (Green et al. 2008a). It is thereforesignificant that the longer incubation times ofQ-CWD prions and altered patterns of PrPSc

deposition in both Tg models were associatedwith an increase in the relative stability of PrPSc

conformers constituting Q-CWD prions.The properties of Q-CWD prions provided

convincing evidence for conformational muta-tions that were suggested, but could not be ob-served, in previous studies of mouse-adaptedscrapie prions in cell cultures treated withswainsonine, an inhibitor of Golgi a-mannosi-dase II (Li et al. 2010). Because the swainsonine-induced properties of cell-derived mouse pri-ons reverted to their original characteristicswhen propagated in vivo, and there were nodetectable differences in the conformations ofPrPSc generated under conditions of swainso-nine treatment compared with untreated cells,claims of strain mutation were indirectly basedon the differing properties of prions fromswainsonine-treated and untreated cells usinga cell panel assay. In contrast, CWD and Q-CWD prions are comprised of distinct PrPSc

conformers that produce discernable pheno-types in two lines of Tg mice.

In accordance with western blot–basedanalyses showing higher conformational stabil-ities of PrPSc produced in the brains of CWD-infected Tg(DeerPrP) mice compared withTg(ElkPrP) mice (Angers et al. 2010), C-CSAconfirmed that the conformation of deer PrPSc

in RK13 cells expressing deer PrP (RKDþ) wasmore stable than elk PrPSc produced during in-fection of Elk-21þ. Because Elk-21þ and RKDþ

propagated CWD prions from the same source,differences in conformational stability resultfrom the effects of residue 226, the sole primarystructural difference between elk and deer PrP.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



WHAT IS THE THREAT OF CWD TOHUMANS?

The identification and characterization of dis-tinct CWD strains, and the influence of PrPprimary structure on their stabilities, is impor-tant when considering the potential for inter-species transmission. The appearance of vCJDfollowing human exposure to BSE (Bruce et al.1997; Hill et al. 1997) raises public healthconcerns about the human species barrier toother animal prion diseases, particularlyCWD. North American hunters harvest thou-sands of deer and elk each year, and it is notcurrently mandatory that these animals are test-ed for CWD; therefore, it is likely that humansconsume CWD prions. Additionally, CWD pri-ons have been shown in the skeletal muscle andfat of deer (Angers et al. 2006; Race et al. 2009b).The substantial market for elk antler velvet intraditional Asian medicine also warrants con-cern (Angers et al. 2009).

Estimates of the zoonotic potential ofCWD are currently mixed. Surveillance current-ly shows no evidence of transmission to humans(Belay et al. 2004; Mawhinney et al. 2006).Although initial cell-free conversion studiessuggested that the ability of CWD prions totransform human PrPC into protease-resistantPrP (PrPSc) was low (Raymond et al. 2000),subsequent results showed that cervid PrPSc in-duced the conversion of human PrPC by PMCA,following CWD prion-strain stabilization bysuccessive passages in vitro or in vivo (Barriaet al. 2011). These results have implicationsfor the human species barrier to CWD andunderscore the role of strain adaptation oninterspecies transmission barriers. Additionalstudies using Tg mice expressing human PrPC

showed that CWD prions failed to induce dis-ease following intracerebral infection (Konget al. 2005; Tamguney et al. 2006; Sandberget al. 2010). However, CWD transmission wasreported in squirrel monkeys (Saimiri sciureus)after intracerebral inoculation (Marsh et al.2005; Race et al. 2009a). Moreover, using RT-QuIC to model the transmission barrier ofCWD to human rPrP, the work of Davenportand colleagues suggests that, at the level of

protein–protein interactions, CWD adapts toa new species more readily than does BSE andthat the barrier preventing transmission ofCWD to humans may be less robust than esti-mated (Davenport et al. 2015).

CONCLUSIONS AND FUTURE PROSPECTS

Some 15 years ago, CWD was perhaps the leastunderstood of all the prion diseases in animalsand humans. Known to be highly contagious,its origins and mode of transmission were un-clear, and it was not known whether multipleCWD strains existed or whether CWD prionsposed a risk to other animals or humans. Themain objectives at that time were to developrapid and sensitive bioassays for CWD prionsand to experimentally address the risks thatCWD prions posed to humans and other spe-cies. Developing Tg mice that were the first re-liable bioassay for rapid and sensitive detectionof CWD prions was a significant advance. Withthese resources in hand, investigators have beenable to obtain important information aboutCWD pathogenesis and the molecular mecha-nisms of prion propagation, species barriers,and strains. Using CWD-susceptible Tg mice,it has become possible to bioassay CWD prionsin tissues, body fluids, and secretions of deerand elk, which have provided insights into themode of transmission of this highly contagiousdisease. The study of intermammalian speciesbarriers in Tg mice allows investigators to mod-el the risks posed to humans and livestock fromexposure to CWD prions, and this informationhelps facilitate management decisions designedto minimize interspecies prion transmission.During this time, the development of morefacile, sensitive approaches to amplify prionsin vitro, such as PMCA and RT-QuIC, have rev-olutionized our ability to detect prions, even atextremely low titers. In concert, cell culturemodels have provided an alternate means ofCWD titration that has largely superseded bio-assay in Tg mice and has provided insights intostrategies for developing compounds that inhib-it CWD propagation. The development of com-pounds such as IND24 provides significantoptimism for treating this currently incurable



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



disease. Finally, studies of CWD using thesenewly developed tools have provided unexpect-ed mechanistic insights into PrPC-to-PrPSc con-version, particularly the role of the b2–a2loop/a-helix 3 epitope and the proposal thatprion strains exist as a continuum of conforma-tional quasispecies.

Looking toward the future, although manyenigmatic issues surrounding the biology ofCWD have been addressed and resolved overthe last decade and a half, substantial questionsremain. First, it is unclear whether managementof this highly contagious, lethal disease will besuccessful in the coming decades. To date, thegeographical areas and numbers of host speciesaffected by CWD have been expanding un-checked. Given the high densities of deer pop-ulations in many areas of North America andEurope, and the persistence of CWD infectivityin the environment, there is little reason tosuspect that this trend will not continue. Thepossibility that CWD will establish a footholdin the large populations of migratory caribou innorthern Canada is of particular concern giventhe reliance of First Nation peoples on these andother cervid species. Beyond North America,expansion of CWD to other countries, as oc-curred in the case of the Republic of Korea,and more recently Norway, is also of impor-tance, although the limited amount of surveil-lance that has been put in place to monitordisease would appear to be at odds with theseconcerns. Although Tg models have been ex-tremely valuable as a means of detecting andstudying CWD prions, they have significantdrawbacks when it comes to reproducing fea-tures of peripheral pathogenesis in the naturalhost. Because this feature most likely influenc-es the highly contagious nature of disease, thedevelopment of tractable model systems inwhich to accurately study the parameters gov-erning peripheral CWD pathogenesis andcontagious transmission in the wild is a highpriority.

Second, our studies suggest that CWD-in-fected deer, elk, or moose produce prions withdifferent biological characteristics. This findingraises several important questions, includingthe evolutionary effects of passage among these

species on the infectious properties of CWDand whether CWD prions arising from thesedifferent sources have varying zoonotic poten-tials. Related to this question, and given theimpact of strain properties on interspeciestransmission, systematically addressing the zoo-notic potential, as well as the tissue distribu-tions of newly recognized CWD strains, includ-ing CWD1 and CWD2 (Angers et al. 2010),would appear to remain high priorities. Thetransmission of BSE to humans in the form ofvCJD underscores the potential and the unpre-dictability of newly emergent prions to causedisease in humans that consume, or are other-wise exposed to, CWD prions. The best that canbe said about the current scientific evidencesurrounding this issue is that the zoonoticpotential of CWD remains uncertain.

REFERENCES

Almberg ES, Cross PC, Johnson CJ, Heisey DM, RichardsBJ. 2011. Modeling routes of chronic wasting diseasetransmission: Environmental prion persistence promotesdeer population decline and extinction. PLoS One 6:e19896.

Angers RC, Browning SR, Seward TS, Sigurdson CJ, MillerMW, Hoover EA, Telling GC. 2006. Prions in skeletalmuscles of deer with chronic wasting disease. Science311: 1117.

Angers RC, Seward TS, Napier D, Green M, Hoover E,Spraker T, O’Rourke K, Balachandran A, Telling GC.2009. Chronic wasting disease prions in elk antler velvet.Emerg Infect Dis 15: 696–703.

Angers RC, Kang HE, Napier D, Browning S, Seward T,Mathiason C, Balachandran A, McKenzie D, Castilla J,Soto C, et al. 2010. Prion strain mutation determinedby prion protein conformational compatibility and pri-mary structure. Science 328: 1154–1158.

Angers R, Christiansen J, Nalls AV, Kang HE, Hunter N,Hoover E, Mathiason CK, Sheetz M, Telling GC. 2014.Structural effects of PrP polymorphisms on intra- andinterspecies prion transmission. Proc Natl Acad Sci 111:11169–11174.

Baeten LA, Powers BE, Jewell JE, Spraker TR, Miller MW.2007. A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi). J Wildl Dis 43: 309–314.

Barret A, Tagliavini F, Forloni G, Bate C, Salmona M, Co-lombo L, De Luigi A, Limido L, Suardi S, Rossi G, et al.2003. Evaluation of quinacrine treatment for priondiseases. J Virol 77: 8462–8469.

Barria MA, Telling GC, Gambetti P, Mastrianni JA, Soto C.2011. Generation of a new form of human PrPSc in vitroby interspecies transmission from cervid prions. J BiolChem 286: 7490–7495.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Bartz JC, Marsh RF, McKenzie DI, Aiken JM. 1998. The hostrange of chronic wasting disease is altered on passage inferrets. Virology 251: 297–301.

Belay ED, Maddox RA, Williams ES, Miller MW, Gambetti P,Schonberger LB. 2004. Chronic wasting disease andpotential transmission to humans. Emerg Infect Dis 10:977–984.

Benestad SL, Mitchell G, Simmons M, Ytrehus B, Vikøren T.2016. First case of chronic wasting disease in Europe in aNorwegian free-ranging reindeer. Vet Res 47: 88.

Berry DB, Lu D, Geva M, Watts JC, Bhardwaj S, Oehler A,Renslo AR, DeArmond SJ, Prusiner SB, Giles K. 2013.Drug resistance confounding prion therapeutics. ProcNatl Acad Sci 110: E4160–E4169.

Berry D, Giles K, Oehler A, Bhardwaj S, DeArmond SJ,Prusiner SB. 2015. Use of a 2-aminothiazole to treatchronic wasting disease in transgenic mice. J Infect Dis212: S17–S25.

Bessen RA, Marsh RF. 1994. Distinct PrP properties suggestthe molecular basis of strain variation in transmissiblemink encephalopathy. J Virol 68: 7859–7868.

Bessen RA, Robinson CJ, Seelig DM, Watschke CP, Lowe D,Shearin H, Martinka S, Babcock AM. 2011. Transmissionof chronic wasting disease identifies a prion strain caus-ing cachexia and heart infection in hamsters. PLoS One 6:e28026.

Bian J, Napier D, Khaychuck V, Angers R, Graham C, TellingG. 2010. Cell-based quantification of chronic wastingdisease prions. J Virol 84: 8322–8326.

Bian J, Kang HE, Telling GC. 2014. Quinacrine promotesreplication and conformational mutation of chronicwasting disease prions. Proc Natl Acad Sci 111: 6028–6033.

Browning SR, Mason GL, Seward T, Green M, Eliason GA,Mathiason C, Miller MW, Williams ES, Hoover E, TellingGC. 2004. Transmission of prions from mule deer and elkwith chronic wasting disease to transgenic mice express-ing cervid PrP. J Virol 78: 13345–13350.

Bruce ME, Will RG, Ironside JW, McConnell I, DrummondD, Suttie A, McCardle L, Chree A, Hope J, Birkett C, et al.1997. Transmissions to mice indicate that “new variant”CJD is caused by the BSE agent. Nature 389: 498–501.

Christen B, Hornemann S, Damberger FF, Wuthrich K.2009. Prion protein NMR structure from tammar wal-laby (Macropus eugenii) shows that the b2–a2 loop ismodulated by long-range sequence effects. J Mol Biol389: 833–845.

Conner MM, Miller MW, Ebinger MR, Burnham KP. 2007.A meta-BACI approach for evaluating management in-tervention on chronic wasting disease in mule deer. EcolAppl 17: 140–153.

Crowell J, Hughson A, Caughey B, Bessen RA. 2015. Hostdeterminants of prion strain diversity independent ofprion protein genotype. J Virol 89: 10427–10441.

Davenport KA, Henderson DM, Bian J, Telling GC, Mathia-son CK, Hoover EA. 2015. Insights into chronic wastingdisease and bovine spongiform encephalopathy speciesbarriers by use of real-time conversion. J Virol 89: 9524–9531.

Di Bari MA, Nonno R, Castilla J, D’Agostino C, Pirisinu L,Riccardi G, Conte M, Richt J, Kunkle R, Langeveld J, et al.

2013. Chronic wasting disease in bank voles: Character-isation of the shortest incubation time model for priondiseases. PLoS Pathog 9: e1003219.

Doh-Ura K, Iwaki T, Caughey B. 2000. Lysosomotropicagents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74: 4894–4897.

Dube C, Mehren KG, Barker IK, Peart BL, Balachandran A.2006. Retrospective investigation of chronic wasting dis-ease of cervids at the Toronto Zoo, 1973–2003. Can Vet J47: 1185–1193.

Dulberger J, Hobbs NT, Swanson HM, Bishop CJ, MillerMW. 2010. Estimating chronic wasting disease effectson mule deer recruitment and population growth. J WildlDis 46: 1086–1095.

Duque Velasquez C, Kim C, Herbst A, Daude N, Garza MC,Wille H, Aiken J, McKenzie D. 2015. Deer prion proteinsmodulate the emergence and adaptation of chronicwasting disease strains. J Virol 89: 12362–12373.

Fasano C, Campana V, Griffiths B, Kelly G, Schiavo G, Zur-zolo C. 2008. Gene expression profile of quinacrine-cured prion-infected mouse neuronal cells. J Neurochem105: 239–250.

Fox KA, Jewell JE, Williams ES, Miller MW. 2006. Patternsof PrPCWD accumulation during the course of chronicwasting disease infection in orally inoculated mule deer(Odocoileus hemionus). J Gen Virol 87: 3451–3461.

Geremia C, Miller MW, Hoeting JA, Antolin MF, Hobbs NT.2015. Bayesian modeling of prion disease dynamics inmule deer using population monitoring and capture-re-capture data. PLoS One 10: e0140687.

Ghaemmaghami S, Watts JC, Nguyen HO, Hayashi S, DeAr-mond SJ, Prusiner SB. 2011. Conformational transfor-mation and selection of synthetic prion strains. J MolBiol 413: 527–542.

Goni F, Mathiason CK, Yim L, Wong K, Hayes-Klug J, NallsA, Peyser D, Estevez V, Denkers N, Xu J, et al. 2015.Mucosal immunization with an attenuated Salmonellavaccine partially protects white-tailed deer from chronicwasting disease. Vaccine 33: 726–733.

Gossert AD, Bonjour S, Lysek DA, Fiorito F, Wuthrich K.2005. Prion protein NMR structures of elk and of mouse/elk hybrids. Proc Natl Acad Sci 102: 646–650.

Green KM, Castilla J, Seward TS, Napier DL, Jewell JE, SotoC, Telling GC. 2008a. Accelerated high fidelity prion am-plification within and across prion species barriers. PLoSPathog 4: e1000139.

Green KM, Browning SR, Seward TS, Jewell JE, Ross DL,Green MA, Williams ES, Hoover EA, Telling GC. 2008b.The elk PRNP codon 132 polymorphism controls cervidand scrapie prion propagation. J Gen Virol 89: 598–608.

Haley NJ, Hoover EA. 2015. Chronic wasting disease of cer-vids: Current knowledge and future perspectives. AnnuRev Anim Biosci 3: 305–325.

Haley NJ, Seelig DM, Zabel MD, Telling GC, Hoover EA.2009a. Detection of CWD prions in urine and saliva ofdeer by transgenic mouse bioassay. PLoS One 4: e4848.

Haley NJ, Mathiason CK, Zabel MD, Telling GC, Hoover EA.2009b. Detection of sub-clinical CWD infection in con-ventional test-negative deer long after oral exposure tourine and feces from CWDþ deer. PLoS One 4: e7990.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Haley NJ, Mathiason CK, Carver S, Zabel M, Telling GC,Hoover EA. 2011. Detection of CWD prions in salivary,urinary, and intestinal tissues of deer: Potential mecha-nisms of prion shedding and transmission. J Virol 85:6309–6318.

Haley NJ, Carver S, Hoon-Hanks LL, Henderson DM, Dav-enport KA, Bunting E, Gray S, Trindle B, Galeota J, LeVanI, et al. 2014. Detection of chronic wasting disease in thelymph nodes of free-ranging cervids by real-time quak-ing-induced conversion. J Clin Microbiol 52: 3237–3243.

Hamir AN, Cutlip RC, Miller JM, Williams ES, Stack MJ,Miller MW, O’Rourke KI, Chaplin MJ. 2001. Preliminaryfindings on the experimental transmission of chronicwasting disease agent of mule deer to cattle. J Vet DiagnInvest 13: 91–96.

Hamir AN, Miller JM, Cutlip RC, Stack MJ, Chaplin MJ,Jenny AL, Williams ES. 2003. Experimental inoculationof scrapie and chronic wasting disease agents in raccoons(Procyon lotor). Vet Rec 153: 121–123.

Hamir AN, Kunkle RA, Cutlip RC, Miller JM, O’Rourke KI,Williams ES, Miller MW, Stack MJ, Chaplin MJ, Richt JA.2005. Experimental transmission of chronic wasting dis-ease agent from mule deer to cattle by the intracerebralroute. J Vet Diagn Invest 17: 276–281.

Hamir AN, Kunkle RA, Miller JM, Greenlee JJ, Richt JA.2006a. Experimental second passage of chronic wastingdisease (CWDmule deer) agent to cattle. J Comp Pathol 134:63–69.

Hamir AN, Kunkle RA, Cutlip RC, Miller JM, Williams ES,Richt JA. 2006b. Transmission of chronic wasting diseaseof mule deer to Suffolk sheep following intracerebralinoculation. J Vet Diagn Invest 18: 558–565.

Hamir AN, Gidlewski T, Spraker TR, Miller JM, CreekmoreL, Crocheck M, Cline T, O’Rourke KI. 2006c. Preliminaryobservations of genetic susceptibility of elk (Cervus ela-phus nelsoni) to chronic wasting disease by experimentaloral inoculation. J Vet Diagn Invest 18: 110–114.

Hamir AN, Miller JM, Kunkle RA, Hall SM, Richt JA. 2007a.Susceptibility of cattle to first-passage intracerebral inoc-ulation with chronic wasting disease agent from white-tailed deer. Vet Pathol 44: 487–493.

Hamir AN, Kunkle RA, Miller JM, Cutlip RC, Richt JA,Kehrli ME Jr, Williams ES. 2007b. Age-related lesions inlaboratory-confined raccoons (Procyon lotor) inoculatedwith the agent of chronic wasting disease of mule deer.J Vet Diagn Invest 19: 680–686.

Hamir AN, Kunkle RA, Nicholson EM, Miller JM, Hall SM,Schoenenbruecher H, Brunelle BW, Richt JA. 2008. Pre-liminary observations on the experimental transmissionof chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer. J Comp Pathol 138: 121–130.

Heaton MP, Leymaster KA, Freking BA, Hawk DA, Smith TP,Keele JW, Snelling WM, Fox JM, Chitko-McKown CG,Laegreid WW. 2003. Prion gene sequence variation with-in diverse groups of U.S. sheep, beef cattle, and deer.Mamm Genome 14: 765–777.

Heisey DM, Mickelsen NA, Schneider JR, Johnson CJ, John-son CJ, Langenberg JA, Bochsler PN, Keane DP, Barr DJ.2010. Chronic wasting disease (CWD) susceptibility ofseveral North American rodents that are sympatric withcervid CWD epidemics. J Virol 84: 210–215.

Henderson DM, Manca M, Haley NJ, Denkers ND, Nalls AV,Mathiason CK, Caughey B, Hoover EA. 2013. Rapidantemortem setection of CWD prions in seer saliva.PLoS One 8: e74377.

Henderson DM, Davenport KA, Haley NJ, Denkers ND,Mathiason CK, Hoover EA. 2015. Quantitative assess-ment of prion infectivity in tissues and body fluids byreal-time quaking-induced conversion. J Gen Virol 96:210–219.

Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, Col-linge J, Doey LJ, Lantos P. 1997. The same prion straincauses vCJD and BSE. Nature 389: 448–450.

Jewell JE, Conner MM, Wolfe LL, Miller MW, Williams ES.2005. Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronicwasting disease. J Gen Virol 86: 2127–2134.

Jewell JE, Brown J, Kreeger T, Williams ES. 2006. Prion pro-tein in cardiac muscle of elk (Cervus elaphus nelsoni) andwhite-tailed deer (Odocoileus virginianus) infected withchronic wasting disease. J Gen Virol 87: 3443–3450.

Johnson C, Johnson J, Clayton M, McKenzie D, Aiken J.2003. Prion protein gene heterogeneity in free-rangingwhite-tailed deer within the chronic wasting disease af-fected region of Wisconsin. J Wildl Dis 39: 576–581.

Johnson CJ, Phillips KE, Schramm PT, McKenzie D, AikenJM, Pedersen JA. 2006a. Prions adhere to soil mineralsand remain infectious. PLoS Pathog 2: e32.

Johnson C, Johnson J, Vanderloo JP, Keane D, Aiken JM,McKenzie D. 2006b. Prion protein polymorphisms inwhite-tailed deer influence susceptibility to chronic wast-ing disease. J Gen Virol 87: 2109–2114.

Johnson CJ, Pedersen JA, Chappell RJ, McKenzie D, AikenJM. 2007. Oral transmissibility of prion disease is en-hanced by binding to soil particles. PLoS Pathog 3: e93.

Joly DO, Ribic CA, Langenberg JA, Beheler K, Batha CA,Dhuey BJ, Rolley RE, Bartelt G, Van Deelen TR, SamuelMD. 2003. Chronic wasting disease in free-ranging Wis-consin white-tailed deer. Emerg Infect Dis 9: 599–601.

Kataoka N, Nishimura M, Horiuchi M, Ishiguro N. 2005.Surveillance of chronic wasting disease in sika deer, Cer-vus nippon, from Tokachi district in Hokkaido. J Vet MedSci 67: 349–351.

Keane DP, Barr DJ, Bochsler PN, Hall SM, Gidlewski T,O’Rourke KI, Spraker TR, Samuel MD. 2008. Chronicwasting disease in a Wisconsin white-tailed deer farm.J Vet Diagn Invest 20: 698–703.

Khan MQ, Sweeting B, Mulligan VK, Arslan PE, CashmanNR, Pai EF, Chakrabartty A. 2010. Prion disease suscep-tibility is affected by b-structure folding propensity andlocal side-chain interactions in PrP. Proc Natl Acad Sci107: 19808–19813.

Kim TY, Shon HJ, Joo YS, Mun UK, Kang KS, Lee YS. 2005.Additional cases of chronic wasting disease in importeddeer in Korea. J Vet Med Sci 67: 753–759.

Klingenstein R, Lober S, Kujala P, Godsave S, Leliveld SR,Gmeiner P, Peters PJ, Korth C. 2006. Tricyclic antidepres-sants, quinacrine and a novel, synthetic chimera thereofclear prions by destabilizing detergent-resistant mem-brane compartments. J Neurochem 98: 748–759.

Klohn PC, Stoltze L, Flechsig E, Enari M, Weissmann C.2003. A quantitative, highly sensitive cell-based infectiv-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



ity assay for mouse scrapie prions. Proc Natl Acad Sci 100:11666–11671.

Kong Q, Huang S, Zou W, Vanegas D, Wang M, Wu D, YuanJ, Zheng M, Bai H, Deng H, et al. 2005. Chronic wastingdisease of elk: Transmissibility to humans examined bytransgenic mouse models. J Neurosci 25: 7944–7949.

Korth C, May BC, Cohen FE, Prusiner SB. 2001. Acridineand phenothiazine derivatives as pharmacotherapeuticsfor prion disease. Proc Natl Acad Sci 98: 9836–9841.

Kreeger TJ, Montgomery DL, Jewell JE, Schultz W, WilliamsES. 2006. Oral transmission of chronic wasting disease incaptive Shira’s moose. J Wildl Dis 42: 640–645.

Kurt TD, Perrott MR, Wilusz CJ, Wilusz J, Supattapone S,Telling GC, Zabel MD, Hoover EA. 2007. Efficient in vitroamplification of chronic wasting disease PrPRES. J Virol81: 9605–9608.

Kurt TD, Telling GC, Zabel MD, Hoover EA. 2009. Trans-species amplification of PrPCWD and correlation withrigid loop 170N. Virology 387: 235–243.

Kurt TD, Bett C, Fernandez-Borges N, Joshi-Barr S, Horne-mann S, Rulicke T, Castilla J, Wuthrich K, Aguzzi A,Sigurdson CJ. 2014. Prion transmission prevented bymodifying the b2–a2 loop structure of host PrPC. J Neu-rosci 34: 1022–1027.

Kurt TD, Jiang L, Fernandez-Borges N, Bett C, Liu J, Yang T,Spraker TR, Castilla J, Eisenberg D, Kong Q, et al. 2015.Human prion protein sequence elements impede cross-species chronic wasting disease transmission. J Clin Invest125: 1485–1496.

LaFauci G, Carp RI, Meeker HC, Ye X, Kim JI, Natelli M,Cedeno M, Petersen RB, Kascsak R, Rubenstein R. 2006.Passage of chronic wasting disease prion into transgenicmice expressing Rocky Mountain elk (Cervus elaphusnelsoni) PrPC. J Gen Virol 87: 3773–3780.

Lee YH, Sohn HJ, Kim MJ, Kim HJ, Park KJ, Lee WY, Yun EI,Tark DS, Choi YP, Cho IS, et al. 2013. Experimentalchronic wasting disease in wild type VM mice. J VetMed Sci 75: 1107–1110.

Li J, Browning S, Mahal SP, Oelschlegel AM, Weissmann C.2010. Darwinian evolution of prions in cell culture. Sci-ence 327: 869–872.

Liberski PP, Guiroy DC, Williams ES, Walis A, Budka H.2001. Deposition patterns of disease-associated prionprotein in captive mule deer brains with chronic wastingdisease. Acta Neuropathol (Berl) 102: 496–500.

Marsh RF, Kincaid AE, Bessen RA, Bartz JC. 2005. Interspe-cies transmission of chronic wasting disease prions tosquirrel monkeys (Saimiri sciureus). J Virol 79: 13794–13796.

Martin S, Jeffrey M, Gonzalez L, Siso S, Reid HW, Steele P,Dagleish MP, Stack MJ, Chaplin MJ, Balachandran A.2009. Immunohistochemical and biochemical character-istics of BSE and CWD in experimentally infectedEuropean red deer (Cervus elaphus elaphus). BMC VetRes 5: 26.

Mathiason CK, Powers JG, Dahmes SJ, Osborn DA, MillerKV, Warren RJ, Mason GL, Hays SA, Hayes-Klug J, SeeligDM, et al. 2006. Infectious prions in the saliva andblood of deer with chronic wasting disease. Science 314:133–136.

Mathiason CK, Nalls AV, Seelig DM, Kraft SL, Carnes K,Anderson KR, Hayes-Klug J, Hoover EA. 2013. Suscep-tibility of domestic cats to chronic wasting disease. J Virol87: 1947–1956.

Mawhinney S, Pape WJ, Forster JE, Anderson CA, Bosque P,Miller MW. 2006. Human prion disease and relative riskassociated with chronic wasting disease. Emerg Infect Dis12: 1527–1535.

Mays CE, Joy S, Li L, Yu L, Genovesi S, West FG, Westaway D.2012. Prion inhibition with multivalent PrPSc bindingcompounds. Biomaterials 33: 6808–6822.

Meade-White K, Race B, Trifilo M, Bossers A, Favara C,Lacasse R, Miller M, Williams E, Oldstone M, Race R,et al. 2007. Resistance to chronic wasting disease in trans-genic mice expressing a naturally occurring allelic variantof deer prion protein. J Virol 81: 4533–4539.

Meyerett C, Michel B, Pulford B, Spraker TR, Nichols TA,Johnson T, Kurt T, Hoover EA, Telling GC, Zabel MD.2008. In vitro strain adaptation of CWD prions by serialprotein misfolding cyclic amplification. Virology 382:267–276.

Miller MW, Williams ES. 2003. Prion disease: Horizontalprion transmission in mule deer. Nature 425: 35–36.

Miller MW, Wild MA, Williams ES. 1998. Epidemiology ofchronic wasting disease in captive Rocky Mountain elk.J Wildl Dis 34: 532–538.

Miller MW, Williams ES, McCarty CW, Spraker TR, KreegerTJ, Larsen CT, Thorne ET. 2000. Epizootiology of chronicwasting disease in free-ranging cervids in Colorado andWyoming. J Wildl Dis 36: 676–690.

Miller MW, Williams ES, Hobbs NT, Wolfe LL. 2004. Envi-ronmental sources of prion transmission in mule deer.Emerg Infect Dis 10: 1003–1006.

Miller MW, Swanson HM, Wolfe LL, Quartarone FG, HuwerSL, Southwick CH, Lukacs PM. 2008. Lions and prionsand deer demise. PLoS ONE 3: e4019.

Mitchell GB, Sigurdson CJ, O’Rourke KI, Algire J, Harring-ton NP, Walther I, Spraker TR, Balachandran A. 2012.Experimental oral transmission of chronic wasting dis-ease to reindeer (Rangifer tarandus tarandus). PLoS ONE7: e39055.

Nalls AV, McNulty E, Powers J, Seelig DM, Hoover C, HaleyNJ, Hayes-Klug J, Anderson K, Stewart P, Goldmann W,Hoover EA, Mathiason CK. 2013. Mother to offspringtransmission of chronic wasting disease in Reeves’ munt-jac deer. PLoS ONE 8: e71844.

Oklahoma Department of Wildlife Conservation. 2002.http://www.wildlifedepartment.com/facts_maps/archives/2002Whitetailarticles.htm.

O’Rourke KI, Besser TE, Miller MW, Cline TF, Spraker TR,Jenny AL, Wild MA, Zebarth GL, Williams ES. 1999. PrPgenotypes of captive and free-ranging Rocky Mountainelk (Cervus elaphus nelsoni) with chronic wasting disease.J Gen Virol, 80: 2765–2769.

O’Rourke KI, Spraker TR, Hamburg LK, Besser TE, BraytonKA, Knowles DP. 2004. Polymorphisms in the prion pre-cursor functional gene but not the pseudogene are asso-ciated with susceptibility to chronic wasting disease inwhite-tailed deer. J Gen Virol 85: 1339–1346.

O’Rourke KI, Spraker TR, Zhuang D, Greenlee JJ, GidlewskiTE, Hamir AN. 2007. Elk with a long incubation prion



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org


http://www.wildlifedepartment.com/facts_maps/archives/2002Whitetailarticles.htm







disease phenotype have a unique PrPd profile. Neurore-port 18: 1935–1938.

Pattison IH. 1965. Experiments with scrapie with specialreference to the nature of the agent and the pathologyof the disease. In Slow, Latent and Temperate Virus Infec-tions, NINDB Monograph No. 2 (ed. Gajdusek DC, GibbsCJ Jr, and Alpers MP), pp. 249–257. U.S. GovernmentPrinting, Washington, DC.

Peretz D, Scott MR, Groth D, Williamson RA, Burton DR,Cohen FE, Prusiner SB. 2001. Strain-specified relativeconformational stability of the scrapie prion protein. Pro-tein Sci 10: 854–863.

Perez DR, Damberger FF, Wuthrich K. 2010. Horse prionprotein NMR structure and comparisons with relatedvariants of the mouse prion protein. J Mol Biol 400:121–128.

Pritzkow S, Morales R, Moda F, Khan U, Telling GC, HooverE, Soto C. 2015. Grass plants bind, retain, uptake, andtransport infectious prions. Cell Rep 11: 1168–1175.

Prusiner SB, Scott M, Foster D, Pan KM, Groth D, MirendaC, Torchia M, Yang SL, Serban D, Carlson GA, et al. 1990.Transgenetic studies implicate interactions between ho-mologous PrP isoforms in scrapie prion replication. Cell63: 673–686.

Pulford B, Spraker TR, Wyckoff AC, Meyerett C, Bender H,Ferguson A, Wyatt B, Lockwood K, Powers J, Telling GC,Wild MA, Zabel MD. 2012. Detection of PrPCWD infeces from naturally exposed Rocky Mountain elk (Cervuselaphus nelsoni) using protein misfolding cyclic amplifi-cation. J Wildl Dis 48: 425–434.

Race BL, Meade-White KD, Ward A, Jewell J, Miller MW,Williams ES, Chesebro B, Race RE. 2007. Levels of abnor-mal prion protein in deer and elk with chronic wastingdisease. Emerg Infect Dis 13: 824–830.

Race B, Meade-White K, Race R, Chesebro B. 2009a. Prioninfectivity in fat of deer with chronic wasting disease. JVirol 83: 9608–9610.

Race B, Meade-White KD, Miller MW, Barbian KD, Ruben-stein R, LaFauci G, Cervenakova L, Favara C, Gardner D,Long D, et al. 2009b. Susceptibilities of nonhuman pri-mates to chronic wasting disease. Emerg Infect Dis 15:1366–1376.

Race B, Meade-White K, Miller MW, Fox KA, Chesebro B.2011. In vivo comparison of chronic wasting disease in-fectivity from deer with variation at prion protein residue96. J Virol 85: 9235–9238.

Race B, Meade-White KD, Phillips K, Striebel J, Race R,Chesebro B. 2014. Chronic wasting disease agents in non-human primates. Emerg Infect Dis 20: 833–837.

Raymond GJ, Bossers A, Raymond LD, O’Rourke KI,McHolland LE, Bryant 3rd PK, Miller MW, WilliamsES, Smits M, Caughey B. 2000. Evidence of a molecularbarrier limiting susceptibility of humans, cattle andsheep to chronic wasting disease. EMBO J 19: 4425–4430.

Raymond GJ, Olsen EA, Lee KS, Raymond LD, Bryant 3rdPK, Baron GS, Caughey WS, Kocisko DA, McHollandLE, Favara C, et al. 2006. Inhibition of protease-resistantprion protein formation in a transformed deer cell lineinfected with chronic wasting disease. J Virol 80: 596–604.

Raymond GJ, Raymond LD, Meade-White KD, HughsonAG, Favara C, Gardner D, Williams ES, Miller MW,Race RE, Caughey B. 2007. Transmission and adaptationof chronic wasting disease to hamsters and transgenicmice: evidence for strains. J Virol 81: 4305–4314.

Rhyan JC, Miller MW, Spraker TR, McCollum M, Nol P,Wolfe LL, Davis TR, Creekmore L, O’Rourke KI. 2011.Failure of fallow deer (Dama dama) to develop chronicwasting disease when exposed to a contaminated envi-ronment and infected mule deer (Odocoileus hemionus). JWildl Dis 47: 739–744.

Roels S, Renard C, De Bosschere H, Geeroms R, Van PouckeM, Peelman L, Vanopdenbosch E. 2004. Detection ofpolymorphisms in the prion protein gene in the Belgiansheep population: some preliminary data. Vet Q 26: 3–11.

Ryou C, Prusiner SB, Legname G. 2003. Cooperative bind-ing of dominant-negative prion protein to kringle do-mains. J Mol Biol 329: 323–333.

Sandberg MK, Al-Doujaily H, Sigurdson CJ, Glatzel M,O’Malley C, Powell C, Asante EA, Linehan JM, BrandnerS, Wadsworth JD, Collinge J. 2010. Chronic wasting dis-ease prions are not transmissible to transgenic mice over-expressing human prion protein. J Gen Virol 91: 2651–2657.

Saunders SE, Bartz JC, Vercauteren KC, Bartelt-Hunt SL.2011. An enzymatic treatment of soil-bound prions ef-fectively inhibits replication. Appl Environ Microbiol 77:4313–4317.

Saunders SE, Bartelt-Hunt SL, Bartz JC. 2012. Occurrence,transmission, and zoonotic potential of chronic wastingdisease. Emerg Infect Dis 18: 369–376.

Schatzl HM, Wopfner F, Gilch S, von Brunn A, Jager G. 1997.Is codon 129 of prion protein polymorphic in humanbeings but not in animals? Lancet 349: 1603–1604.

Schettler E, Steinbach F, Eschenbacher-Kaps I, Gerst K,Muessdoerffer F, Risch K, Streich WJ, Frolich K. 2006.Surveillance for prion disease in cervids, Germany. EmergInfect Dis 12: 319–322.

Scott M, Groth D, Foster D, Torchia M, Yang SL, DeArmondSJ, Prusiner SB. 1993. Propagation of prions with artifi-cial properties in transgenic mice expressing chimericPrP genes. Cell 73: 979–988.

Seelig DM, Nalls AV, Flasik M, Frank V, Eaton S, MathiasonCK, Hoover EA. 2015. Lesion profiling and subcellularprion localization of cervid chronic wasting disease indomestic cats. Vet Pathol 52: 107–119.

Seidel B, Thomzig A, Buschmann A, Groschup MH, PetersR, Beekes M, Terytze K. 2007. Scrapie agent (Strain 263K)can transmit disease via the oral route after persistence insoil over years. PLoS ONE 2: e435.

Sigurdson CJ, Williams ES, Miller MW, Spraker TR,O’Rourke KI, Hoover EA. 1999. Oral transmission andearly lymphoid tropism of chronic wasting disease PrPresin mule deer fawns (Odocoileus hemionus). J Gen Virol 80:2757–2764.

Sigurdson CJ, Spraker TR, Miller MW, Oesch B, Hoover EA.2001. PrPCWD in the myenteric plexus, vagosympathetictrunk and endocrine glands of deer with chronic wastingdisease. J Gen Virol 82: 2327–2334.

Sigurdson CJ, Barillas-Mury C, Miller MW, Oesch B, vanKeulen LJ, Langeveld JP, Hoover EA. 2002. PrPCWD lym-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



phoid cell targets in early and advanced chronic wastingdisease of mule deer. J Gen Virol 83: 2617–2628.

Sigurdson CJ, Manco G, Schwarz P, Liberski P, Hoover EA,Hornemann S, Polymenidou M, Miller MW, Glatzel M,Aguzzi A. 2006. Strain fidelity of chronic wasting diseaseupon murine adaptation. J Virol 80: 12303–12311.

Sigurdson CJ, Mathiason CK, Perrott MR, Eliason GA,Spraker TR, Glatzel M, Manco G, Bartz JC, Miller MW,Hoover EA. 2008. Experimental chronic wasting disease(CWD) in the ferret. J Comp Pathol 138: 189–196.

Sigurdson CJ, Nilsson KP, Hornemann S, Heikenwalder M,Manco G, Schwarz P, Ott D, Rulicke T, Liberski PP, JuliusC, et al. 2009. De novo generation of a transmissiblespongiform encephalopathy by mouse transgenesis.Proc Natl Acad Sci 106: 304–309.

Sigurdson CJ, Nilsson KP, Hornemann S, Manco G, Fernan-dez-Borges N, Schwarz P, Castilla J, Wuthrich K, AguzziA. 2010. A molecular switch controls interspecies priondisease transmission in mice. J Clin Invest 120: 2590–2599.

Smith CB, Booth CJ, Pedersen JA. 2011. Fate of prions insoil: A review. J Environ Qual 40: 449–461.

Sohn HJ, Kim JH, Choi KS, Nah JJ, Joo YS, Jean YH, AhnSW, Kim OK, Kim DY, Balachandran A. 2002. A case ofchronic wasting disease in an elk imported to Korea fromCanada. J Vet Med Sci 64: 855–858.

Spraker TR, Miller MW, Williams ES, Getzy DM, Adrian WJ,Schoonveld GG, Spowart RA, O’Rourke KI, Miller JM,Merz PA. 1997. Spongiform encephalopathy in free-rang-ing mule deer (Odocoileus hemionus), white-tailed deer(Odocoileus virginianus), and Rocky Mountain elk (Cer-vus elaphus nelsoni) in northcentral Colorado. J Wildl Dis33: 1–6.

Tamguney G, Giles K, Bouzamondo-Bernstein E, Bosque PJ,Miller MW, Safar J, DeArmond SJ, Prusiner SB. 2006.Transmission of elk and deer prions to transgenic mice.J Virol 80: 9104–9114.

Tamguney G, Miller MW, Wolfe LL, Sirochman TM, Glid-den DV, Palmer C, Lemus A, DeArmond SJ, Prusiner SB.2009. Asymptomatic deer excrete infectious prions infaeces. Nature 461: 529–532.

Telling GC. 2011. Transgenic mouse models and prionstrains. Top Curr Chem 305: 79–99.

Telling G. 2013. Chronic wasting disease and the develop-ment of research models. In Prions and diseases: Volume 2,Animals, humans and the environment (ed. Zou W-Q,Gambetti P), pp 45–57. Springer, New York.

Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M,Cohen FE, DeArmond SJ, Prusiner SB. 1995. Prion prop-agation in mice expressing human and chimeric PrP

transgenes implicates the interaction of cellular PrPwith another protein. Cell 83: 79–90.

Telling GC, Parchi P, DeArmond SJ, Cortelli P, Montagna P,Gabizon R, Mastrianni J, Lugaresi E, Gambetti P, PrusinerSB. 1996. Evidence for the conformation of the patho-logic isoform of the prion protein enciphering and prop-agating prion diversity. Science 274: 2079–2082.

U.S. Census Bureau. 2011. National Survey of Fishing, Hunt-ing, and Wildlife-Associated Recreation (FHWAR). www.census.gov/prod/www/fishing.html.

Vickery CM, Lockey R, Holder TM, Thorne L, Beck KE,Wilson C, Denyer M, Sheehan J, Marsh S, Webb PR,et al. 2014. Assessing the susceptibility of transgenicmice overexpressing deer prion protein to bovine spon-giform encephalopathy. J Virol 88: 1830–1833.

Vilette D, Andreoletti O, Archer F, Madelaine MF, Vilotte JL,Lehmann S, Laude H. 2001. Ex vivo propagation of in-fectious sheep scrapie agent in heterologous epithelialcells expressing ovine prion protein. Proc Natl Acad Sci98: 4055–4059.

Wadsworth JD, Asante EA, Desbruslais M, Linehan JM,Joiner S, Gowland I, Welch J, Stone L, Lloyd SE, HillAF, et al. 2004. Human prion protein with valine 129prevents expression of variant CJD phenotype. Science306: 1793–1796.

Wasserberg G, Osnas EE, Rolley RE, Samuel MD. 2009. Hostculling as an adaptive management tool for chronic wast-ing disease in white-tailed deer: A modelling study. J ApplEcol 46: 457–466.

Williams ES. 2003. Scrapie and chronic wasting disease. ClinLab Med 23: 139–159.

Williams ES. 2005. Chronic wasting disease. Vet Pathol 42:530–549.

Williams ES, Miller MW. 2002. Chronic wasting disease indeer and elk in North America. Rev Sci Tech 21: 305–316.

Williams ES, Young S. 1980. Chronic wasting disease ofcaptive mule deer: A spongiform encephalopathy. J WildlDis 16: 89–98.

Williams ES, Young S. 1982. Spongiform encephalopathy ofRocky Mountain elk. J Wildl Dis 18: 465–471.

Williams ES, Young S. 1992. Spongiform encephalopathiesin Cervidae. Rev Sci Tech Off Int Epiz 11: 551–567.

Williams ES, Young S. 1993. Neuropathology of chronicwasting disease of mule deer (Odocoileus hemionus) andelk (Cervus elaphus nelsoni). Vet Pathol 30: 36–45.

Wolfe LL, Fox KA, Miller MW. 2014. “Atypical” chronicwasting disease in PRNP genotype 225FF mule deer. JWildl Dis 50: 660–665.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org


http://www.census.gov/prod/www/fishing.html





published online February 13, 2017Cold Spring Harb Perspect Med Julie A. Moreno and Glenn C. Telling Molecular Mechanisms of Chronic Wasting Disease Prion Propagation

Subject Collection Prion Diseases

-Synuclein: Multiple System Atrophy Prionsα

Aoyagi, et al.Amanda L. Woerman, Joel C. Watts, Atsushi Disease Prion Propagation

Molecular Mechanisms of Chronic Wasting

Julie A. Moreno and Glenn C. TellingGenetics of Synucleinopathies

Robert L. NussbaumGenetics of Amyotrophic Lateral Sclerosis

Mehdi Ghasemi and Robert H. Brown, Jr.

Alzheimer's Disease-Amyloid Prions and the Pathobiology ofβ

Joel C. Watts and Stanley B. Prusiner

ExpansionsC9orf72The Genetics of

BroeckhovenIlse Gijselinck, Marc Cruts and Christine Van

Expansions RepeatC9ORF72Disease Mechanisms of

Tania F. Gendron and Leonard Petrucelli

TDP-43 PrionsTakashi Nonaka and Masato Hasegawa

-SynucleinαCell Biology and Pathophysiology of

SüdhofJacqueline Burré, Manu Sharma and Thomas C. Polyglutamine-Containing Proteins

Prion-Like Characteristics of

Margaret M.P. Pearce and Ron R. Kopito

Symptom Onset Explained by Tau Propagation?Chronic Traumatic Encephalopathy: Is Latency in

McKeeJoshua Kriegel, Zachary Papadopoulos and Ann C.

HomeostasisTherapeutic Strategies for Restoring Tau

GestwickiZapporah T. Young, Sue Ann Mok and Jason E.

Cross-Seeding, and TransmissionNoncerebral Amyloidoses: Aspects on Seeding,

Katarzyna Lundmark, et al.Gunilla T. Westermark, Marcus Fändrich,

Neurodegenerative DiseaseFused in Sarcoma Neuropathology in

Ian R.A. Mackenzie and Manuela Neumann

ComplexesStructural and Chemical Biology of Presenilin

Pettersson, et al.Douglas S. Johnson, Yue-Ming Li, Martin

DiseasesExperimental Models of Inherited PrP Prion

Joel C. Watts and Stanley B. Prusiner

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved


http://perspectivesinmedicine.cshlp.org/cgi/collection/

http://perspectivesinmedicine.cshlp.org/cgi/collection/


molecular mechanisms of chronic wasting disease prion...

Documents