Strain Fidelity of Chronic Wasting Disease upon Murine Adaptation

Chronic wasting disease (CWD), a prion disease of deer and elk,is highly prevalent in some regions of North America. The establishmentof mouse-adapted CWD prions has proven difficult due to thestrong species barrier between mice and deer. Here we reportthe efficient transmission of CWD to transgenic mice overexpressingmurine PrP. All mice developed disease 500 ± 62 daysafter intracerebral CWD challenge. The incubation period decreasedto 228 ± 103 days on secondary passage and to 162 ±6 days on tertiary passage. Mice developed very large, radiallystructured cerebral amyloid plaques similar to those of CWD-infecteddeer and elk. PrP^Sc was detected in spleen, indicating thatmurine CWD was lymphotropic. PrP^Sc glycoform profiles maintaineda predominantly diglycosylated PrP pattern, as seen with CWDin deer and elk, across all passages. Therefore, all pathological,biochemical, and histological strain characteristics of CWDappear to persist upon repetitive serial passage through mice.These findings indicate that the salient strain-specific propertiesof CWD are encoded by agent-intrinsic components rather thanby host factors.

INTRODUCTION

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Introduction

Mammalian prion diseases are believed to be caused by the misfoldingof a host-encoded cellular protein, PrP^C, into an aggregated,beta-sheet-rich, insoluble isoform, PrP^Sc, which self-propagatesand leads to fatal neurodegeneration (25, 38). Insoluble PrP^Scaggregates are detectable in the central nervous system andskeletal muscle and also throughout the lymphoid system in asubset of diseases, including variant (22, 47) and sporadic(19) Creutzfeldt-Jakob disease in humans, scrapie in sheep andgoats (21), and chronic wasting disease (CWD) in cervids (34,43).

CWD is a geographically widespread and locally prevalent priondisease of North American cervids. CWD occurs naturally in wildand captive mule deer, white-tailed deer, Rocky Mountain elk(51), and wild moose (M. W. Miller, unpublished data). The originsof CWD and its relationship to other prion diseases remain uncertain(33). Mouse-adapted prion strains of sheep scrapie and bovinespongiform encephalopathy (BSE) have proven very useful forstudying and comparing prion strain properties (7, 28) and forunderstanding the peripheral pathogenesis of prion disease (11,23, 29, 30, 37). However, the transmission of CWD to wild-typemice is inefficient (12) in contrast to transgenic mice expressingthe deer or elk PrP sequences (4, 12, 24). This suggests thata species barrier exists between cervids and mice. Here we useda transgenic mouse model that overexpresses murine PrP to developa murine-adapted CWD strain of prion. We report that this straininduces a unique histopathological phenotype and displays biochemicaland biophysical properties similar to those of deer CWD butdistinct from those of a Rocky Mountain Laboratory (RML) strainof mouse-adapted scrapie. These strain-specific properties werestable over three generations of serial transmission in mice.These newly generated murine CWD prions provide a tool for furtherstudying the biophysical nature of prion strains, e.g., by identifyingdifferential PrP^Sc-interacting proteins.

MATERIALS AND METHODS

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Materials and Methods

Tga20 mice and prion infection. Transgenic mice overexpressing murine PrP (17) were intracerebrallyinoculated in the left parietal cortex with 30 µl of a10% brain homogenate (5% for subsequent passages) from a terminal,CWD-infected mule deer or an uninfected control deer (mock).Mice were monitored every second day, and scrapie was diagnosedaccording to clinical criteria, including ataxia, kyphosis,and hind leg paresis. Mice were sacrificed at the onset of terminaldisease. Mice were maintained under specific pathogen-free conditions,and all experiments were performed in accordance with the animalwelfare guidelines of the Kanton of Zürich.

Histochemical and immunohistochemical stains. Tissues were fixed in 4% buffered formalin, treated with 98%formic acid for 1 h, and postfixed for $≥$ 24 h prior to paraffinembedding. Two-micrometer-thick sections were cut on positivelycharged glass slides and stained with hematoxylin and eosinor immunostained using antibodies for PrP (SAF84), astrocytes(glial fibrillary acidic protein [GFAP]), or microglia (Iba1).For PrP staining, sections were deparaffinized and incubatedfor 6 min in 98% formic acid and then washed in distilled waterfor 5 min. Sections were heated to 100°C in citrate buffer(pH 6.0), cooled for 3 min, and then washed in distilled waterfor 5 min. Immunohistochemical stains were performed on an automatedNexus staining apparatus (Ventana Medical Systems) using anIVIEW DAB detection kit (Ventana). After incubation with protease1 (Ventana) for 16 min, sections were incubated with anti-PrPSAF-84 (SPI bio; 1:200) for 32 min. Sections were counterstainedwith hematoxylin. GFAP immunohistochemistry for astrocytes (1:1,000for 24 min; DAKO) and Iba1 (1:2,500 for 32 min; Wako Chemicals)for microglia were similarly performed, however, with antigenretrieval by heating to 100°C in EDTA buffer (pH 8.0). Histoblotanalyses were performed as previously described (45).

For thioflavin T and Congo red staining, frozen brain sectionswere fixed in 70% ethanol for 10 min. For thioflavin staining,sections were immersed in 1% thioflavin T for 3 min, followedby 1% acetic acid for 10 min and then distilled water for 5min. For Congo red staining, slides were immersed in an alkalinesolution and then stained with Congo red solution for 20 min.

Lesion profile. Twelve anatomic brain regions were selected in accordance withprevious strain typing analysis protocols (13, 18) by usingthree to five mice per passage. Spongiosis was evaluated ona scale of 0 to 5 (not detectable, mild, moderate, severe, andstatus spongiosis). Gliosis and PrP^Sc content were scored ona scale of 0 to 3 (not detectable, mild, moderate, and severe).A sum of the three scores resulted in the value that was employedin order to obtain the lesion profile for the individual animal.Histological analysis was performed by independent investigatorsblinded to animal identification.

Ultrastructural characterization. Small samples (1 mm³) were retrieved from formalin-fixed, paraffin-embeddedblocks and reversed to electron microscopy as previously described(3). Grids stained with lead citrate and uranyl acetate wereobserved and photographed with a JEM 100C transmission electronmicroscope at 80 kV.

Western blots. Ten-percent brain or spleen homogenates were prepared in phosphate-bufferedsaline using a ribolyzer, and 80 to 90 µg protein wasdiluted with Tris lysis buffer (10 mM Tris, 10 mM EDTA, 100mM NaCl, 0.5% NP-40, and 0.5% deoxycholate) and digested with100 µg/ml proteinase K for 30 min at 37°C. Sodiumdodecyl sulfate-based loading buffer was then added, and sampleswere heated at 95°C for 5 min prior to electrophoresis througha 12% Bis-Tris precast gel (Invitrogen), followed by transferto a nitrocellulose membrane by wet blotting. Proteins weredetected with the following panel of "POM" anti-PrP antibodies(36): POM1 (epitope amino acids [aa] 121 to 230), POM2 (epitopeaa 58 to 64, 66 to 72, 74 to 80, and 82 to 88), POM3 (epitopeaa 95 to 100), and POM11 (epitope aa 64 to 72 and 72 to 80).Signals were visualized with an ECL detection kit (Pierce).

RESULTS

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Results

Incubation period and clinical disease. tga20 transgenic mice overexpress mouse PrP owing to a Prnp-encodingvector known as the "half-genomic construct," which is basedon a murine PrP minigene lacking intron 2 (17). tga20 mice wereinoculated intracerebrally with CWD from a terminally infectedmule deer. All inoculated mice developed neurologic diseasewith an incubation period (IP) of 500 ± 62 (range, 408to 521) days postinoculation (Fig. 1A). Remarkably, the clinicalattack rate was 100% (10/10). This contrasts with the poor infectibilityof wild-type mice with CWD and may be attributed to the higherPrP^C expression levels in tga20 mice.

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FIG. 1. Survival of tga20 PrP-overexpressing mice challenged with CWD prions. (A) First passage of mule deer CWD performed on two separate occasions shows similar survival curves. Four mice were used for subpassage into additional tga20 mice (indicated by ${alpha}$ , ß, ${gamma}$ , and ${delta}$ ). Mock-inoculated mice were periodically sacrificed at the time points indicated. (B) Second passage of CWD-infected brains from four mice. Mouse ${varepsilon}$ was used for the third subpassage. (C) Third passage of CWD. An RML scrapie survival curve is shown for comparison.

To determine whether the new murine CWD strain would adapt furtherin the mouse, we performed a second passage of CWD. This wasaccomplished by injecting brain homogenate from CWD-infected,terminally sick mice into another group of tga20 mice. The secondpassage led to an abbreviated IP: mice developed terminal diseasewith an IP of 228 ± 103 dpi (range, 139 to 438) (Fig.1B). By the third passage, the incubation period dropped to162 days, with little variability among the mice (±6days) (Fig. 1C). In contrast, tga20 mice inoculated with 3 x10³ 50% lethal dose units of RML scrapie developed terminalneurologic disease after 74 days (±5 days) (Fig. 1C).

Clinical signs were similar among mice receiving primary deerCWD and murine-adapted CWD and included kyphosis, ataxia, tremors,and intense pruritus (Fig. 2 and see the supplemental material).CWD-infected mice often showed a clinical phenotype of obsessivepruritus. Mice continued scratching, even with palliative careof open abrasions, making euthanasia unavoidable. Exogenouscauses of ulceration were not evident in the skin; no ectoparasitesor other infectious agents were observed.

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FIG. 2. Clinical signs, genetics, and structural biology of murine CWD. (A) Mice with clinical signs of CWD are characterized by kyphosis, pruritus, ataxia, and tremors in contrast to mice with RML scrapie which are primarily ataxic with stiff tails (arrowhead). The CWD-infected mouse is scratching (arrow indicates skin abrasion). (B) A PrP gene sequence alignment for the mouse and mule deer shows 90.2% homology. Amino acids are color coded as follows: red, small and hydrophobic; blue, acidic; magenta, basic; green, hydroxyl and amine and basic. (C and D) Superposition of the mean nuclear magnetic resonance (NMR) structures of the polypeptide segment of aa 125 to 229 (C) and 165 to 172 (D) in ePrP(121-230) (red) and mPrP(121-230) (blue). A spline function was drawn through the C positions. The radius of the cylindrical rods representing the polypeptide chains is proportional to the mean backbone displacement per residue (XX), as evaluated after superposition for best fit of the atoms N, C, and C' in the 20 energy-minimized conformers used to represent the NMR structures (9). The PrP structure from the CWD source deer used herein may vary slightly from the elk.

Genetic and structural considerations. The prion sequence of the mule deer was compared to that frommouse. When comparing the sequences from residues 23 to 231,there was a 90.2% sequence homology between the deer and themouse (Fig. 2B). A comparison of the three-dimensional structuresof the elk and mouse prion proteins shows that both proteinshave the same globular fold, consisting of a flexibly disordereddomain of $~$ 100 residues and a C-terminal domain of similar size,with three ${alpha}$ -helices and a short antiparallel ß-sheet(20). However, the loop region that connects the second ßstrand with the second ${alpha}$ -helix is very precisely defined in theelk prion protein but disordered in mouse PrP^C (Fig. 2C and D).Elk and mule deer amino acid sequences are identical at thisloop region.

Disease phenotype and lesion profiles. We performed a detailed investigation of the histopathologiclesions in each of the serial passages of CWD (Fig. 3A). Spongiformlesions were most prominent in the neuopil of thalamus, hippocampus,and basal ganglia. Some cases also displayed extensive damageto the cerebral cortex and the white matter tracts. Lesionsappeared to acquire greater severity with each serial passage(Fig. 3A). By the third passage, mice developed a subtotal electiveloss of cerebellar granular cells, a finding absent in the firstand second passages (Fig. 3B). Astrogliosis and microglial activationwere often severe and regionally extensive in CWD-infected mice(Fig. 3A). The findings described above were quantified usinga standardized lesion-profiling methodology (35) and indicatedthat the severity of spongiform degeneration, astrogliosis,and PrP^Sc deposition increased with each serial passage (Fig.3C). Some mock-inoculated animals had mild-to-moderate vacuolizationof white matter tracts, but no neuronal loss or extensive astrogliosis,microgliosis, or plaques.

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FIG. 3. Histopathology and PrP^Sc plaques in CWD-infected mice. (A) Brain at hippocampus. PrP plaques are visible in hematoxylin and eosin (HE) stains and by immunohistochemical labeling for PrP, particularly in the corpus callosum. Extensive astrocytosis and microglial activation is evident. Note the increased severity of gliosis, microglial activation, and plaque deposition with each passage. Bar = 200 µm. (B) Severe degeneration with massive loss of the cerebellar granular cells occurred by the third passage. Bar = 200 µm or 20 µm. (C) Lesion profile of CWD infected mice, comparing first through third passage. Profile scores are determined by spongiform change, gliosis, and PrP^Sc deposits in the specific brain regions indicated. (D) Vascular plaques in meninges and brain parenchyma of CWD-infected mice. Bar = 50 µm.

Four of nine (44%) first-passage, CWD-inoculated mice had multiplelarge (50- to 300-µm) plaques visible in the hematoxylin-and-eosin-stainedsections, which immunolabeled for PrP. Plaques appeared in thebasal ganglia, thalamus, and hippocampus and separated whitematter fiber tracts in the corpus callosum and cerebellum. Extensiveplaques are not a feature of RML mouse scrapie (A. Aguzzi andC. J. Sigurdson, unpublished data) but are common with cervidCWD (5, 44). Remarkably, PrP plaques and deposits were oftenperivascular (Fig. 3D) as in CWD in deer and elk but were alsowithin vessel walls. However, a subset of mice developed onlysmall, diffuse PrP deposits typical of RML mouse scrapie ordeveloped both plaques and diffuse deposits (Fig. 4A). Thirdpassage of the "plaque" strain led to plaque formation in allinfected mice. Plaques were composed of dense deposits similarto those seen with the first passage.

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FIG. 4. Plaque characterization in CWD-infected mice. (A) PrP^Sc deposits vary from small, diffuse aggregates to dense plaques, often occurring as a mixture of both deposit types. Bar = 50 µm. (B) Plaques were thioflavin positive and showed birefringence after Congo red staining indicative of amyloid formation. Bar = 20 µm.

Plaque structure. By light microscopy, murine CWD plaques were composed of fiberbundles emanating from a dense core in stellate arrangementand were unicentric or multicentric. Thioflavin T positivityand Congo red birefringence indicated that the plaques had ahighly organized amyloid fibril cross-ß fold structure(Fig. 4B). By electron microscopy, plaques were composed ofcompact amyloid bundles and appeared as subpial (Fig. 5A), unicentric(Fig. 5B and E), or perivascular deposits (Fig. 5D). Overall,the morphologies of plaques recapitulated that observed in muledeer with CWD (27) and were similar to the human prion diseaseGerstmann-Straussler-Scheinker disease (GSS). As with GSS, microglialcells were in close contact with the amyloid bundles (Fig. 5C)(6, 26).

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FIG. 5. Ultrastructural analysis of murine CWD plaques. (A) Subpial deposits were composed of numerous unicentric plaques. Magnification, x2,000. (B) Unicentric kuru-like plaque. Magnification, x6,000. (C) A plaque margin showing microglial cells. Amyloid bundles are in close contact with the cell (arrow). Magnification, x13,000. (D) Perivascular plaque. The nucleus (arrow) and cytoplasm (arrowhead) of the pericyte is visible. Magnification, x8,300. (E) Unicentric plaque. Magnification, x8,300. (F) High-power view of separate amyloid bundles. Magnification, x20,000.

Histoblot analysis. To visualize proteinase K (PK)-resistant PrP^Sc in brain andspleen, we performed histoblot analyses of frozen tissue sectionspressed onto nitrocellulose, proteinase K treated, and labeledwith anti-PrP antibodies. Here again we found abundant PrP^Scplaques and diffuse deposits in the brain in a pattern thatvaried from those of RML mice (Fig. 6A). Mice inoculated withsecond and third passages of the "plaque" strain developed PrP^Scdeposits in the spleen in follicular patterns similar to thoseof RML-infected mice (Fig. 6B).

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FIG. 6. Histoblots of brain and spleen reveal differences in neural PrP^Sc distribution in murine CWD (overall less PrP^Sc) compared to RML mouse scrapie. Abundant PrP^Sc deposits were present in spleen of some CWD-infected mice. No PrP^Sc stain was seen in the mock-inoculated mouse. Bar = 500 µm.

Biochemical profile of murine CWD. We analyzed proteinase K-resistant PrP^Sc in brain infected withmurine CWD by Western blotting and compared it with that ofa standard murine RML scrapie isolate and with that of CWD indeer. We found that CWD in deer was characterized by a predominantdiglycosylated isoform of PrP^Sc as described previously (39).In contrast, RML murine scrapie presents with a dominant monoglycosylatedPrP^Sc isoform. In most CWD-infected mice with the "plaque" strain,the diglycosylate-rich glycoform pattern was faithfully propagated.However, two of seven mice developed nearly equal levels ofdi- and monoglycosylated isoforms on the third passage (Fig.7). Murine first-passage strains of sheep scrapie and BSE (alsoin tga20) showed glycoform ratios similar to that found withthe third passage of murine CWD (Fig. 7). The PK cutting sitein murine CWD was then defined by differential antibody immunoreactivityof PrP^27–30 to be between amino acids 73 to 82, as POM11(epitope aa 64 to 72 and 72 to 80) did not recognize PrP^27–30(data not shown).

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FIG. 7. Biochemical characterization of PrP^Sc in murine CWD (A and B). Western blots and triplot of murine CWD-infected brain showing a predominant diglycosylated band in most mice on first passage. By the third passage, the glycoform pattern shifted slightly with an increase in the monoglycosylated PrP and was similar to deer CWD, murine BSE, and murine sheep scrapie, but unlike the mouse-adapted RML. –, absence of; +, presence of.

DISCUSSION

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Discussion

Prion transmission between species is often inefficient andgoes along with prolonged incubation periods. However, serialpassages within the new species often lead to a shortening ofthe incubation period (48), a phenomenon that has been termedadaptation. Neither the molecular underpinnings of the speciesbarrier nor those of prion adaptation are fully understood.The primary amino acid sequence of host PrP^C clearly contributesto the species barrier effect (40, 50), yet additional factorsintrinsic to the infectious agent, be they PrP^Sc conformation,PrP^Sc aggregational state, and/or ancillary molecules (49),also affect susceptibility and disease phenotype (15, 16, 46).Because of such agent-autonomous factors, species barriers arespecified by the pathogenic prions in addition to the genotypeof the hosts: bovine spongiform encephalopathy is, e.g., readilytransmissible to species that are hardly susceptible to sheepscrapie (32).

Although Prnp is very well conserved among most species, themouse and deer PrP amino acid sequences are relatively divergent.Amino acid identity is only 90.2% between residues 23 to 231,and the respective PrP^C structures strikingly diverge betweenamino acids 165 to 172 (20). This finding may underlie the inefficientCWD prion conversion in wild-type mice. However, even a highdegree of homology between PrP molecules is not predictive forefficient conversion, as single-amino-acid substitutions cancreate a strong species barrier (31).

Despite the structural constraints listed above, we found thatoverexpression of murine PrP of four- to sixfold sufficed toenable the efficient transmission of deer CWD to mice. Thiswas unexpected since the expression levels of Prnp were notfound to be rate limiting for ecotropic prion replication inother systems (14). In contrast, these findings suggest thathost PrP^C expression levels may significantly affect the robustnessof species barriers.

CWD manifested itself in tga20 mice with features that are remarkablysimilar to those of CWD in deer and strikingly different fromthose of the commonly used RML strain of mouse-adapted sheepprions. Some CWD-infected mice developed very large multicentricextracellular amyloid plaques that were intensely stained bythioflavin and Congo red, often positioned within white mattertracts or around vessels, similar to those described for deerwith CWD (44) and for humans with GSS (26). In contrast, plaqueswere never seen in RML-infected tga20 mice or RML-infected wild-typeC57BL/6 inbred, 129Sv inbred, or C57BL/6 x 129Sv crossbred mice(A. Aguzzi, unpublished data).

The plaques were reminiscent of those shown by Scott et al.in mice expressing bovine PrP and challenged with BSE or sheepscrapie prions (41). In mice with plaques, the plaque phenotypewas stable and persisted even after the third serial passageof CWD in mice.

The second passage of CWD in the tga20 mice revealed broadlydivergent disease phenotypes. First, the plaque phenotype wasnot present in all second-passage mice, as a subset of fourmice (inoculated with isolates from a mouse that did not haveplaques) developed only diffuse granular PrP stain depositsas seen by immunohistochemistry, and PrP^Sc signals in the brainby Western blotting was low. Second, one group of second-passagemice developed disease after a remarkably long incubation period.Third, there was also an inconsistent detection of splenic PrP^Scin the second-passage mice. These findings suggest that theremay be multiple CWD strains present in the deer, with each maintaininga presence in the tga20 mice. Alternatively, it is also possiblethat the variability is intrinsically linked to the mechanismof strain adaptation, with the strain phenotype diversifyingas a process of the adaptation.

We considered the possibility that the above variability mayhave resulted from the contamination of the inoculum with ourRML laboratory strain. This, however, is extremely unlikely,as the incubation periods for RML are much shorter than thosereported here. When exposed to limiting infectious doses ofRML, tga20 mice consistently develop disease in <180 days(10, 42) or resist infection completely. In addition, the clinicalsigns for RML differ strikingly from those detected with murineCWD. Finally, the time lag between the first clinical signsand terminal disease is only a few days in RML-infected tga20mice, whereas it extended over weeks in CWD-infected mice.

Clues to prion strain differences lie in the conformationalstate of the misfolded prion protein and in the ratio of thePrP glycoforms (7, 8). Several techniques were employed to explorethese features, including the quantitation of the relative proportionof the three glycoforms by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and determination of the proteinase K cleavagesite (8) with a panel of anti-PrP antibodies recognizing variousepitopes amino- and carboxy-proximal to the putative cleavagesite (36, 53). We found that the murine CWD glycoform profileshifted slightly with serial passage from a highly abundantdiglycosylated PrP^Sc on first passage to an almost equal ratioof di- and monoglycosylated PrP^Sc by the third passage, a ratiowhich was close to that of CWD in deer, murine BSE, and murinesheep scrapie and varied from that of RML. The PK cleavage sitewas assessed in the third passage of murine CWD and localizedto amino acids 73 to 82. This is consistent with the PK-cuttingsites for elk CWD at Gly78 and Gly82 (52) and suggests thatfeatures of the CWD PrP^Sc structure are maintained in the mouse.

CWD prions were serially passaged three times. With each passage,the incubation period decreased and the brain lesions and PrPplaques developed to a greater severity. This is consistentwith other instances of strain adaptation, in which prion virulencetended to increase with time. However, the biochemical, thehistopathological, and even the clinical characteristics ofthe disease remained remarkably constant, suggesting that thephenotype-specifying information is enciphered more within theagent rather than in the host. The stability of the phenotypictraits over three passages suggests that the phenotype-specifyinginformation undergoes replication along with the infectiousprinciple.

Within the framework of the protein-only hypothesis, the abovephenomenon might be explained by postulating that CWD prionspossess a particular steric conformation that is unique, distinctfrom that of RML scrapie prions, and capable of imparting thesame conformation onto mouse PrP^C, which will then propagatethis unique conformation indefinitely upon serial passage (1).

Alternatively, PrP^Sc aggregates derived from CWD-infected deermay be packed in a specific quaternary assembly state that growsappositionally in a growth pattern similar to that of a crystal.Such quasicrystals might be shaped differently from RML aggregatesand might incorporate murine PrP^Sc upon passaging to mice (2).Eventually, repeated serial passages would lead to the propagationof an aggregated moiety that consists exclusively of murinePrP^Sc but retains the unique quaternary structure of CWD prions.

A further model predicates that conventional replicative molecules,such as DNA, RNA, or micro-RNA, might be incorporated into PrP^Sc.While prion infectivity is solely specified by PrP^Sc (the "apoprion"),the strain-specific characteristics would be propagated by such"coprions" (49).

Finally, CWD prions may incorporate specific subsets of hostmolecules, be they proteins, lipids, nucleic acids, or otherconstituents, according to a unique stoichiometry. Prion replicationupon cross-species passage might reproduce this stoichiometry,thereby preserving the strain-specific traits. The propagationof a well-characterized strain of CWD prions within geneticallyhomogeneous hosts yields a powerful tool for testing each ofthe above hypotheses. A particularly promising avenue of futurework consists of utilizing contemporary proteomic tools, suchas differential mass spectrometry with isotope-coded affinitytags, to enumerate non-PrP^Sc components that are associatedwith mouse-passaged CWD prions and may contribute to their uniquephenotypic traits.

ACKNOWLEDGMENTS

We thank M. Heikenwälder and P. Nilsson for critical reviewsof the manuscript and F. Heppner for optimization of the microglialimmunohistochemical stain. We gratefully acknowledge the animalcare staff and the histopathology technical support at the Universityof Zurich.

This study was supported by grants from the European Union (Apopisand TSEUR), the Swiss National Foundation, the National CompetenceCenter for Research on Neural Plasticity and Repair (to A.A.),the National Institutes of Health (K08-AI01802 to C.J.S.), theFoundation for Research at the Medical Faculty, University ofZurich (to C.J.S.), and the United States National Prion ResearchProgram (to C.J.S. and A.A.).

FOOTNOTES

* Corresponding author. Mailing address: UniversitätsSpital Zürich, Institute of Neuropathology, Department of Pathology, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland. Phone: 41 (1) 255 2107. Fax: 41 (1) 255 4402. E-mail: adriano.aguzzi{at}usz.ch.

Published ahead of print on 4 October 2006.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: University Medical Center Hamburg-Eppendorf,Institute of Neuropathology, Martinistrasse 52, D-20246 Hamburg,Germany.

REFERENCES

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