North American and Norwegian Chronic Wasting Disease Prions Exhibit Different Potential for Interspecies Transmission and Zoonotic Risk

Abstract

Background

Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting various species of wild and captive cervids. The risk that CWD poses to cohabiting animals or more importantly to humans is largely unknown.

Methods

In this study, we investigated differences in the capacity of CWD isolates obtained from 6 different cervid species to induce prion conversion in vitro by protein misfolding cyclic amplification. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at expenses of normal prion proteins from various mammals and human, respectively.

Results

Our data suggest that reindeer and red deer from Norway could be the most transmissible CWD prions to other mammals, whereas North American CWD prions were more prone to generate human prions in vitro.

Conclusions

Our results suggest that Norway and North American CWD prions correspond to different strains with distinct spillover and zoonotic potentials.

We investigated in vitro spillover and zoonotic potential of CWD from various cervid species. Our results suggest that Norway CWD prions have a higher potential to infect other animals, but North American CWD appears more prone to generate human prions.

Transmissible spongiform encephalopathies, also called prion diseases, are a group of neurodegenerative disorders affecting various species of mammals, including humans [1]. Creutzfeldt-Jakob disease (CJD) is the most common prion disease in humans; the main animal diseases are scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) affecting cattle, and chronic wasting disease (CWD) in cervids.

Chronic wasting disease is a highly contagious disease found in several species of captive and free-ranging cervids (eg, whitetail deer, mule deer, elk, sika, reindeer, moose, red deer, and muntjac) [2]. The origin of CWD as well as the mechanisms explaining their rapid transmission are not completely understood. Chronic wasting disease has been rapidly expanding geographically and now affects 26 states in United States, 3 Canadian provinces, South Korea, and Northern Europe (Norway, Sweden, and Finland) [3–5]. The risk of transmission of CWD to other animal species (herein referred to as spillover potential) or to humans (herein referred to as zoonotic potential) is currently largely unknown [2]. Several studies have been done to analyze the potential for CWD prions to infect humans [6–13]. However, CWD zoonotic potential is still unclear, mostly because of limitations with animal models.

The infectious agent in prion diseases is composed of a misfolded and aggregated form of the prion protein (termed PrP^Sc), which replicates by the autocatalytic conversion of the normal prion protein (PrP^C) [14]. Despite its simple composition, prions exhibit complex characteristics in common with conventional infectious agents [15]. For example, prions are known to exist in multiple strains that can result in markedly distinct phenotypes as defined by differences in infectivity titer, clinical signs, neuropathological lesion profile, and PrP^Sc properties [16]. It is widely thought that prion strains are caused by PrP^Sc adopting different structures, which can faithfully propagate in vivo and in vitro [17]. In the case of CWD, it is not yet known how many prion strains may occur naturally, although some reports have provided evidence for multiple CWD strains [18, 19]. Also akin to conventional infectious organisms is the ability of different prions to infect only certain species, a characteristic known as “species barrier,” which depends on the degree of sequence similarity of the prion protein between the donor and the host and the specific strain properties of the transmissible agent [20, 21]. As PrP^Sc is transmitted from one species to another, differences in prion protein sequence may lead to new conformations that may manifest as different strains [20–22]. Many prion-susceptible animal species share habitats with cervids and are therefore candidates for CWD infection [2]. Transmission of CWD may result in new animal diseases with the theoretical possibility to have higher zoonotic potential. Experimental infection of several large animals have shown that CWD prions can indeed be transmissible to other animal species [23–26]. However, it is currently unknown whether different CWD strains may have distinct ability to infect other animal species or humans.

In this study, we evaluated differences in the spillover and zoonotic potential of CWD prions derived from 3 different species of North American cervids (whitetail deer, mule deer, and elk) and 3 cervids species from Norway (reindeer, red deer, and moose). For this purpose, we performed in vitro studies of the conversion of PrP^C from different animal species (mouse, hamsters, sheep, cattle, pig, and human) triggered by exposure to different CWD PrP^Sc isolates. We selected these species for our studies because they may plausibly come in contact with CWD-infected cervids or CWD prions shed into the environment.

In vitro conversion studies were done by the protein misfolding cyclic amplification (PMCA) technology, which mimics the process of prion replication that occur in the brain of infected individuals [27]. Many studies have shown that PMCA produces highly transmissible prions that faithfully retain the strain properties [28–33]. PMCA has also been shown to generally reproduce the species barrier and strain adaptation process [33–35], enabling it to be used as a proxy for investigating the transmissibility of prions across species and the strain characteristics of prions generated upon interspecies conversion.

METHODS

Study Populations

For these studies, cortical brain material from CWD-infected whitetail deer (Odocoileus virginianus), elk (Cervus canadensis), and mule deer (Odocoileus hemionus) were selected as species native to North America (sample IDs 813, BD-2, and Db99, respectively). We also used CWD-infected moose (Alces alces), reindeer (Rangifer tarandus), and red deer (Cervus elaphus) (sample IDs 17-4-CD11399, 17-4-CD11087, and 17-4-CD14051, respectively) as Norwegian cervids. For Norway CWD, we used a pool of several brain regions per animal.

Normalization of Chronic Wasting Disease Inoculum

Brain material from CWD-infected cervids was homogenized at 10% (w/v) in phosphate-buffered saline supplemented with complete protease inhibitor. Homogenates were digested with proteinase K (PK) (100 µg/mL) for 1 hour at 37°C with shaking (500 rpm). Digested samples were analyzed by Western blotting with 6H4 primary antibody (1:5000 dilution) and signal was visualized by chemiluminescence. Due to their relatively higher PrP^Sc content, North American CWD samples were normalized to that of Norwegian moose. Norwegian reindeer and red deer were not diluted further because they had relatively less PrP^Sc than Norwegian moose. Normalization was performed using ImageJ software. Dilution factor was calculated based on mean signal intensity (after subtracting background signal) multiplied by the area. Dilution factors of 5.9-, 21.7-, and 3.3-fold were determined for whitetail deer, elk, and mule deer. Dilutions were performed in knock-in deer [36] substrate to preserve concentration of other components in the brain.

Protein Misfolding Cyclic Amplification Procedure

Eleven microliters of normalized CWD brain homogenate (BH) was mixed with 99 µL of each respective substrate BH to create the first 10-fold dilution, which was considered as the 10^–2 dilution. Subsequent dilutions were made by mixing 11 µL from the previous dilution into 99 µL of new BHBH substrate to reach a 10^–9 dilution. Before PMCA, the 10 % BH was supplemented with 6 mM EDTA, 0.05 % digitonin, and 100 µg/µL heparin; these reaction conditions were held constant across each PMCA experiment. With the exception of wild-type mouse and hamster, the other brains were harvested from transgenic mice or gene targeted knock-in mice, selectively bred to harbor the PRNP gene specific to the mammal species of interest (Supplementary Table 1). The 10% BHBH was prepared in conversion buffer (1× phosphate-buffer saline, 150 mM sodium chloride, 1% Triton X-100) with the addition of protease inhibitors. PMCA was conducted within a QSonica700 sonicator at amplitude 20-35A and subjected to 1 round of PMCA (144 sonication/incubation cycles) or 2 rounds (144 and 96 sonication/incubation cycles) in the case of CWD transmission to human. For PMCA passage to a second round, an aliquot of 10 µL from each reaction tube was passaged to a new reaction tube with 90 µL freshly supplemented 10% BH. All PMCA experiments were performed in the same sonicator and included a positive amplification control homologous to the substrate in examination and negative controls without inoculum.

Western Blot

Before blotting, PMCA products were subjected to PK digestion (100 µg/mL; Sigma-Aldrich) for 1 hour at 37°C with shaking at 600 rpm using an Eppendorf thermomixer. After SDS-PAGE and transfer to a Hybond-ECL nitrocellulose membrane (0.45 µm), the membrane was probed overnight with the 6D11 antiprion antibody (used at 1:30 000 dilution; BioLegend). The membrane was treated with ECL solution (Amersham) and viewed under the ChemiDoc XRS image analysis system (Bio-Rad).

Radar Plots

To determine the extent of seed amplification in non-homologous substrates, PrPSc signal in Western blots was considered positive when higher that a threshold of 30× (for zoonotic potential) or 40× (for spillover potential) the standard deviation of the background, calculated using ImageJ software. This threshold was calculated for each membrane as intensity of background varied. The extent of positive amplification in non-homologous substrates was plotted as radar plots.

Estimation of Spillover Potential Index and Zoonotic Potential Index

To compare the capacity of different CWD isolates to convert non-homologous PrP^C of other mammalian species, we created a spillover potential index. Each CWD isolate was assigned a score for each substrate based on the extent of amplification in that substrate. When no positive signal was present at the lowest dilution used (10^–2), a score of zero was assigned. A positive signal at 10^–2 was given a score of 1, and an additional “point” was assigned for each successive positive PMCA signal. The amplification scores for each respective substrate tested were summed to create a spillover potential index, which, in broad terms, represents the propensity of each CWD to potentially cross the species barrier into other mammals.

To estimate the zoonotic potential, we used the second round of PMCA in both 129M and 129V human PrP substrate. Amplification scores were assigned as described above. Values for both M and V substrates were summed to create the final zoonotic potential index.

RESULTS

We evaluated the in vitro spillover and zoonotic potential of CWD prions derived from 3 different species of North American cervids (whitetail deer, mule deer, and elk) and 3 cervid species from Norway (reindeer, red deer, and moose) (see Supplementary Figure 1 for description of the procedures). First, we analyzed the presence and relative quantity of PrP^Sc signal after PK digestion in BHs from each animal (Figure 1). Protease-resistant PrP^Sc signal was higher in the North American CWD material compared with the samples derived from Norway. This may be due to differences in infectivity stage and/or brain region obtained. It is interesting to note that the migration profile for PrP^Sc from Norway CWD-infected animals was different from the profile observed for infected animals in North America (Figure 1). In particular, the protease-resistant fragment from moose CWD showed a markedly smaller size compared with the North American CWD proteins, confirming a previous report [37].

Figure 1.

Comparison of chronic wasting disease from North American and Norway species of deer. Cervid species of North American or Norwegian origin were assessed for brain PrP^Sc content by Western blotting. Ten percent BHs were digested with proteinase K at 100 μg/mL at 37°C for 1 hour. Dilutions were performed into 10% BH from white-tail deer PrP knock-in mouse to preserve the mass of other proteins present in the BH. Samples were denatured and separated by SDS-PAGE using MOPS running buffer and then transferred onto nitrocellulose membrane. After blocking with 2% nonfat milk, 6H4 anti-PrP antibody (1:10000) was applied overnight and followed by antimouse secondary antibody (1:3000). Signal was visualized by chemiluminescence. NBH was not digested with PK and is used as a migration marker for PrP^C.

To normalize the amount of PrP^Sc in different inocula, we estimated the quantity of material present in each sample by densitometric analysis of the Western blots. Samples were normalized for PrP^Sc quantity by diluting the more concentrated samples into BH from gene-targeted knock-in transgenic mice expressing deer PrP. As a result, each inoculum contains a similar amount of PrP^Sc as evaluated by Western blotting (Supplementary Figure 2). First, we investigated the ability of each inoculum to convert deer and elk PrP^C by PMCA. The last dilution of material still capable to sustain detectable PrP^Sc formation in vitro was similar across all samples, except for the conversion of elk PrP^C by Norway moose CWD (Figure 2). Indeed, whereas all inocula showed detectable PrP^Sc signal until a 10^–9 dilution, moose extract converted elk PrP^C only up to 10^–4 dilution.

Figure 2.

Conversion of deer and elk PrP^C by various chronic wasting disease (CWD) isolates. The propensity of each of CWD isolates under study to convert deer (A) and elk (B) PrP^C was assessed by protein misfolding cyclic amplification (PMCA) assay. For this purpose, we used as substrate 10% w/v BH from gene-targeted transgenic mice expressing the deer or elk prion protein [36]. Serial dilutions from 10^–2 to 10^–9 were done to analyze the efficiency of prion amplification in these 2 cervid species, as described in Methods. The PMCA was performed in a water bath sonicator (Qsonica) for 3 days or approximately 144 cycles as described in Methods. After PMCA, samples were treated with proteinase K at 100 μg/mL and analyzed by Western blots using 6D11 (BioLegend) anti-PrP antibody at 1:30000 concentration. NBH was not digested with PK and is used as a migration marker for PrPC. Neg, refers to samples incubated without addition of prions.

Next, we studied the potential of different CWD prions to induce the conversion of PrP^C from various animal species susceptible to prions, including sheep, hamster, mouse, cattle, and pig (Figure 3). The results showed differences in conversion efficiency depending on both the source of PrP^C used as substrate and the CWD inoculum. Positive control experiments using each of these substrates with PrP^Sc from the homologous species showed a high efficiency of prion conversion (Supplementary Figure 3). The signals observed in Figure 3 do not correspond to the inoculum, because when the same samples were analyzed before PMCA, no signal was detected for any of them (Supplementary Figure 4). For generation of sheep prions, whitetail deer and reindeer CWD appeared to be the best (Figure 3A). A lower conversion ability was observed for red deer, and very poor conversion was seen with mule deer, elk, and moose (Figure 3A). In this study, we used ARQ polymorphism as sheep substrate and cannot rule out that using other polymorphisms may impact the results. PrP^C from hamster seems to be the protein most proficiently converted by CWD, with red deer being the best inoculum, followed by reindeer, moose, and whitetail deer (Figure 3B). In contrast, mouse was not a good substrate for in vitro conversion, with all inocula showing a similarly low efficiency and signal detected up to a 10^–3 dilution for all CWD BHs (Figure 3C). Transmission of CWD to cattle is particularly important because bovine prions have been shown to infect humans [38, 39]. Whereas moose, and to a lower extent reindeer, showed a relatively efficient conversion of bovine PrP^C, the other CWD inocula were not able to generate bovine PrP^Sc (Figure 3D). Finally, porcine PrP^C was not converted by any of the CWD inocula during a first round of PMCA (Figure 3E). These results confirm a previous study showing that pig is inherently resistant to prion infection [40].

Figure 3.

Spillover potential of chronic wasting diseases (CWDs) from North American or Norwegian cervids. The ability of cervid PrP^Sc to convert PrP^C from other animal species was investigated by protein misfolding cyclic amplification (PMCA) using BHBH from various mammalian species as a substrate. Experiment was similar as in Figure 2, but using 10% NBH from (A) sheep transgenic mice (ARQ polymorphism), (B) hamster, (C) mice, (D) bovine transgenic mice, (E) pig transgenic mice. Serial 10-fold dilutions were performed to create dilutions up to 10^–9. Samples, with the exception of the NBH migration control, were digested with proteinase K at 100 μg/mL and detected by Western blot using 6D11 (BioLegend) anti-PrP antibody. Markers on the left represent 25 and 20 kDa molecular weight.

To obtain a clear view of the differences in the spillover potential, we made radar plots with the last dilution of CWD BH that was able to generate PrP^Sc in a given species (Figure 4). Signal was considered positive when it was higher than a threshold of 40× the standard deviation of the background for each blot. The shape, orientation, and area of the geometric figures generated by joining all the points enabled us to visually observe differences in the ability of distinct prions to propagate in vitro at the expense of different PrP^C. The shape of the figures depends on the relative position in the radar plot of the different substrates tested, and thus for a comparison it is necessary to put all species in the same place of the plot.

Figure 4.

Spillover potential for chronic wasting disease (CWD) from various species of North American and Norway animals. The propensity of various CWDs to convert PrP^C from different mammalian substrates during protein misfolding cyclic amplification (PMCA) was displayed as radar plots. Signals were determined positive if they surpassed a threshold of 40 times the standard deviation of the Western blot background signal. Numerical scale in each radar plot represents log-fold dilution. When no positive signal was present at the lowest dilution used (10^–2), a score of zero was assigned. A positive signal at 10^–2 was given a score of 1 and an additional “point” was assigned for each successive positive PMCA signal.

To produce a quantitative index of the spillover potential of distinct CWD isolates, we assigned a score for each CWD isolate in each substrate based on the extent of amplification in that substrate, measured as the last brain dilution detectable (see Methods). The amplification scores were summed to create a spillover potential index (Table 1). Our findings suggest that the recently detected CWD prions in Norway have a higher potential to cross species barriers to other mammals than North American CWD prions. Reindeer and red deer seem to be the most transmissible CWD prions, and whitetail deer followed them as the most transmissible CWD in North America (Table 1).

Table 1.

Spillover and Zoonotic Potential Scores for Each CWD-Infected Cervid Species

Cervid Species	Spillover Potential Scorea	Zoonotic Potential Scoreb (Score in 129M + Score in 129V)
Whitetail Deer	9	6 (2 + 4)
Elk	3	4 (2 + 2)
Mule Deer	6	5 (3 + 2)
Red Deer	8	0
Reindeer	12	0
Moose	9	0

Abbreviations: CWD, chronic wasting disease; PMCA, protein misfolding cyclic amplification.

^aSpillover potential score was measured after 1 PMCA round.

^bZoonotic potential score was measured after 2 PMCA rounds.

The cervid-human species barrier is perhaps the most important and has been extensively studied using various in vitro and in vivo assays [6–13, 41]. To investigate the ability of CWD prions to convert human PrP^C (in vitro zoonotic potential), we performed 2 serial rounds of amplification cycles. The reason for analyzing more PMCA rounds for zoonotic potential studies compared with spillover potential was that it is estimated that the species barrier for cervid/human is stronger than that exhibited for other animal species. Because polymorphic variation at residue 129 is known to control susceptibility of humans to various prion diseases [42], amplifications were performed using human PrP with either M or V at residue 129. The results shown in Figure 5 show that none of the CWD inocula were able to convert human PrP^C in a first round of PMCA, indicating a large species barrier. By contrast, BSE and sheep-adapted BSE converted human PrP^C under similar conditions to produce human PrP^Sc with similar characteristics as variant CJD [13]. By comparing the conversion efficiency of CWD in a second PMCA round, we observed that CWD prions from North American cervids were able to successfully convert human PrP^C (Figure 5). The best results were obtained with whitetail deer, mule deer, and to a lower extent with elk. On the contrary, none of the Norway CWD isolates showed any significant conversion of human PrP^C (Figure 5). Table 1 shows the estimated zoonotic potential index for each CWD isolate. The results suggest that North American CWD might represent a larger zoonotic risk than Norway CWD (Table 1). Human PrP^C with V at position 129 appears to be a slightly better substrate for CWD conversion, as judged from radar plots showed in Figure 6.

Figure 5.

Evaluation of potential transmission of North American (A) or Norway (B) chronic wasting disease (CWD) prions to human by protein misfolding cyclic amplification (PMCA). To evaluate the zoonotic potential of CWD to humans, we analyzed PrP^Sc generation with more amplification cycles by performing 2-rounds of serial PMCA (R1 and R2) to further assess the transmission risk from the different CWD isolates. Aliquots of BH from each of the different CWD animals under study were incubated with BH from transgenic mice expressing M or V human PrP^C. All PMCA samples were subjected to a 100 µg/µL proteinase K (PK) digestion before Western blot. Negative control (neg) of either TgHu129M or 129V were included to control for seeding-specific amplification and rule out cross-contamination. For PMCA positive amplification controls (+), either variant CJD (vCJD) for TgHu129M or sporadic CJD (sCJD VV2) for 129V was utilized. Each membrane was probed with the 6D11 primary antibody and NBH (non-PK-digested TgHu129M or TgHu129V) is included to serve as a migration control. WTD, whitetail deer.

Figure 6.

Radar plots for zoonotic potential. Radar plots were prepared using the second round of protein misfolding cyclic amplification (PMCA) in both TgHu129M and TgHu129V substrates. Signals from Figure 5 were deemed positive if they surpassed 30× the standard deviation of the background signal. The last dilution detectable in the second PMCA round was used in the radar plot. Numerical scale represents log-fold dilution of the initial 10% BH seed. As in Figure 4, when no positive signal was present at the lowest dilution used (10^–2), a score of zero was assigned. A positive signal at 10^–2 was given a score of 1 and an additional “point” was assigned for each successive positive PMCA signal. WTD, white-tail deer.

DISCUSSION

Chronic wasting disease is presently propagating uncontrollably among cervids in North America and has been recently detected in geographically distant locations around the world (South Korea, Norway, Sweden, and Finland) [3, 4]. The mechanism by which CWD propagates so efficiently and the number and properties of different natural CWD strains in existence are mostly unknown. It is well established that many animal species known to be susceptible to transmissible prions share habitats with cervids (eg, sheep, cows, rodents, swine, felines, etc) and therefore are potential candidates for infection by CWD prions [2]. Transmission of CWD to other species may result in new forms of prions leading to novel animal diseases with enhanced zoonotic potential, as was reported earlier for the infection of sheep and goats with BSE [43]. The possibility that CWD prions infect other animals is supported by experimental infection of CWD into various species of mammals, including cows, raccoons, and pigs [23–26]. Nevertheless, a comprehensive study of the spillover potential of CWD from different species of cervids in distinct geographic locations has not been done.

In this study, we used the PMCA technology to estimate the potential of various CWD isolates to convert PrP^C from several animal species. We believe this strategy may represent an important tool for characterizing new prion strains for their predicted spillover and zoonotic potentials. This in vitro assessment does not intend to replace infectivity bioassays, but it produces complementary information that can be obtained much faster than time- and effort-consuming animal experiments. PMCA has been widely shown to reproduce in vitro the prion replication process. Indeed PMCA produces PrP^Sc with high levels of infectivity [28–30] and has been shown to faithfully maintain the strain characteristics of the PrP^Sc used as inoculum [31, 32]. In addition, PMCA was able to reproduce the cross-species transmission and prion adaptation processes [33–35]. However, it is important to highlight that PMCA does not include all factors and variables that may have an impact on in vivo prion infection. For example, PMCA studies do not take into account the effect of biological clearance of the infectious agent, bioavailability problems, selective neuronal vulnerability, etc. Furthermore, because a limited number of samples were analyzed, it is possible that isolates obtained from different animals may behave differently. Therefore, the findings obtained in this study need to be taken cautiously.

In our experiments, we evaluated the ability of PrP^Sc from 6 different cervid species, coming either from North America or Norway to replicate at expenses of PrP^C with sequences from diverse mammals, including cattle, sheep, pig, hamster, and mouse. Our findings showed that the conversion efficiency was substantially different depending on both the source of PrP^Sc inoculum and PrP^C substrate. In general terms, Norway CWD appeared more prone to cross species barriers than North American isolates, except for whitetail deer that seemed also highly transmissible (Figures 3 and 4). We estimated a spillover potential index to quantitatively evaluate the differences on transmission potential among distinct CWD prions. Using this approach we found that the index was reindeer > moose = whitetail deer > red deer > mule deer > elk (Table 1). In other words, reindeer seems to be the CWD strain most prone to transmit to other species, whereas elk has the lowest potential for interspecies transmission. We propose that studies of the spillover potential estimated in vitro by PMCA may also be useful to differentiate prion strains. In this sense, our findings suggest that Norwegian CWD prions correspond to different strains from North American CWD prions, supporting previous studies analyzing the PrP^Sc electrophoretic profile and transmission into rodents [37, 44].

The current evidence for CWD transmission to humans is controversial; indeed, although transgenic mice expressing human PrP did not develop disease when challenged with CWD prions in various laboratories [6–8, 41], experimental inoculation of CWD into squirrel monkeys produced disease [9, 10]. Studies in macaques, which are phylogenetically closer to humans than squirrel monkeys [45], have shown mixed results. A study from Czub et al [46] found that CWD prions can induce disease and pathologic abnormalities typical of prion disease in macaques exposed to CWD prions, even by oral inoculation of muscle tissue from cervids affected by CWD. However, a different study found no evidence for prion disease in macaques inoculated with CWD [47]. To assess the cervid/human species barrier, we previously used PMCA to determine prion replication in vitro. We found that, after stabilization by successive passages in deer PrP^C, PrP^Sc from CWD-infected deer can convert human PrP^C into a novel form of PrP^Sc [13]. Our current study to evaluate in vitro zoonotic potential of various CWD prions showed that although the cervid/human barrier is large, we were able to observe generation of human PrP^Sc with some specific CWD strains in a second round of PMCA (Figure 5). The 3 North American CWD isolates were able to sustain generation of human PrP^Sc, with whitetail deer showing the highest efficiency. In contrast, none of the 3 Norway CWD isolates generated any detectable PrP^Sc signal up to the second round of PMCA.

Our data suggest that North American CWD prions might pose a greater risk to humans than the infected animals in Northern Europe. This might be due to Norwegian CWD being less stable prion strains compared with North American CWD, which have had longer time to replicate in cervids and become stabilized through many rounds of natural infection. Our findings may also provide important information to understand the diversity of natural CWD prion strains in different animals across distinct geographical areas and their consequences for the spillover into other animal species, including humans.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. This study was funded in part by a grant from the National Institute of Health (P01AI077774; to C. S. and G. C. T.). The characterization of the Norwegian cervids was performed in and supported by the Norwegian Veterinary Institute (Project Number 12081) and Norwegian Environment Agency (Project Number 3201040004).

Potential conflicts of interest. C. S. is the inventor on several patents related to the protein misfolding cyclic amplification (PMCA) technology and is currently Founder, Chief Scientific Officer, and member of the Board of Directors of Amprion Inc., a biotech company focusing on the commercial utilization of PMCA for prion diagnosis. S. P. also has a conflict of interest related to the PMCA technology and Amprion. Finally, The University of Texas System has licensed intellectual property to Amprion. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.