Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice
Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice
Jifeng Bian, Jeffrey R. Christiansen, Julie A. Moreno, Sarah J. Kane, Vadim Khaychuk, Joseph Gallegos, Sehun Kim, and Glenn C. Telling PNAS first published May 30, 2019 https://doi.org/10.1073/pnas.1903947116 Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved May 7, 2019 (received for review March 7, 2019)
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Significance
Our gene-targeting strategy addresses several critical issues relating to chronic wasting disease (CWD), a contagious, lethal neurodegenerative prion disease affecting wild as well as captive cervids, which poses an uncertain risk to humans. First, we show that residue 226, the sole primary structural difference between deer and elk prion protein, dictates the selection and propagation of different CWD strains. Because the strain properties of prions affect their host-range potential, these findings suggest that CWD from elk and deer pose distinct risks to humans and other species. Second, we show that these gene-targeted mice offer an unprecedented means to address distinctive aspects of CWD peripheral pathogenesis and horizontal transmission that are not afforded by previously generated transgenic mouse models.
Abstract
Although the unifying hallmark of prion diseases is CNS neurodegeneration caused by conformational corruption of host prion protein (PrP) to its infective counterpart, contagious transmission of chronic wasting disease (CWD) results from shedding of prions produced at high titers in the periphery of diseased cervids. While deer and elk PrP primary structures are equivalent except at residue 226, which is glutamate in elk and glutamine in deer, the effect of this difference on CWD pathogenesis is largely unknown. Using a gene-targeting approach where the mouse PrP coding sequence was replaced with elk or deer PrP, we show that the resulting GtE226 and GtQ226 mice had distinct kinetics of disease onset, prion conformations, and distributions of prions in the brains of diseased mice following intracerebral CWD challenge. These findings indicate that amino acid differences at PrP residue 226 dictate the selection and propagation of divergent strains in deer and elk with CWD. Because prion strain properties largely dictate host-range potential, our findings suggest that prion strains from elk and deer pose distinct risks to sympatric species or humans exposed to CWD. GtE226 and GtQ226 mice were also highly susceptible to CWD prions following intraperitoneal and oral exposures, a characteristic that stood in stark contrast to previously produced transgenic models. Remarkably, disease transmission was effective when infected mice were cohoused with naïve cagemates. Our findings indicate that gene-targeted mice provide unprecedented opportunities to accurately investigate CWD peripheral pathogenesis, CWD strains, and mechanisms of horizontal CWD transmission.
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Discussion Notwithstanding the considerable advantages of Tg mouse models for studying prions, including the capacity to decrease incubation times by transgene overexpression (13), the inability to control random integration of transgene arrays of variable copy number at undefined genomic locations complicates the interpretation of certain analyses, in particular comparative studies designed to assess the effects of PrP primary structural differences on prion pathogenesis. Here we describe the production and initial characterization of two Gt mouse models expressing the PrP coding sequences of elk and deer, which differ only at residue 226. We draw three overarching conclusions from our studies: first, replacement of the coding sequence for mouse PrP with that of deer or elk PrP renders the resulting Gt mice susceptible to CWD prions; second, amino acid variation at residue 226 of deer and elk PrP controls the tempo of disease onset, as well as the targeted accumulation and conformational properties of the resulting prions, reflecting the section and propagation of distinct cervid prion strains; and third, Gt mice offer an unprecedented means to address distinctive aspects of peripheral CWD pathogenesis and contagious transmission that were not afforded by previous Tg mouse models.
Gt Mice Are Susceptible to Cervid Prions. Our findings detailing uniform susceptibility, and relatively rapid responses of GtE226+/+ and GtQ226+/+ mice to elk and deer CWD prions, stand in contrast to the behaviors of previously produced gene targeted mice designed to abrogate species barriers to bovine and human prions. While expression of bovine PrPC conferred susceptibility to BSE prions in Tg mice, even when the level of bovine PrPC expression was lower than that normally found in the brains of cattle (29⇓–31), counterpart Gt mice were paradoxically less susceptible to BSE than wild-type mice (32, 33). Similarly, despite repeated examples of high susceptibility and rapid incubation times of human prions in Tg mice, even at wild-type levels of human PrPC expression (14, 34⇓–36), counterpart Gt mice exhibited longer vCJD prion incubation times compared with wild-type mice (32), as well as prolonged incubation periods and incomplete attack rates in response to sporadic CJD prions (37). The discrepant susceptibilities of these Tg mice and their Gt equivalents were puzzling because they appeared to undermine the primacy of PrP sequence homology in the control of prion transmission barriers (38). In contrast, the differences we observed in incubation times between CWD-susceptible Tg and Gt mice were surprisingly modest. When the effects of residue 226 on the kinetics of disease onset are considered, our findings in Tg and Gt mice expressing cervid PrP confirm the longstanding inverse relationship between levels of CNS PrPC expression and prion incubation times following intracerebral challenge (13).
Our findings and conclusions make it difficult to reconcile the poor responses of previously produced Gt mice to bovine and human prions on the basis of transgene expression alone, and force consideration of several alternate possibilities. First, the likelihood that the relatively facile responses of GtE226 and GtQ226 mice to prion infection are reflective of unusually virulent properties of CWD prions seems remote given that Gt mice were also equally susceptible to cervid-adapted sheep SSBP/1 and cervid-adapted mouse RML prions (Table 1). Second, previously produced Gt mice were generated on a 129 mouse background (32, 37), while the Gt mice reported here were generated using FVB. Whether the discrepant properties of Gt mice result from the effects of mouse genetic backgrounds can be resolved by studying the outcomes of inbreeding Gt mice onto a 129 background, and correspondingly by repeated backcrossing of bovine and human PrP Gt mice with FVB mice. Third, while GtE226 and GtQ226 mice express elk and deer PrP coding sequences, respectively, previously produced Gt mice were engineered to express chimeric mouse–bovine and mouse–human coding sequences, where the signal peptides of the expressed transgenes originated from mouse PrP (32, 37, 47). While such arrangements are not expected to affect the primary structures of mature bovine and human PrP after signal peptidase cleavage and processing, it is nonetheless well established that amino acid substitutions in signal peptide sequences have a considerable impact on PrP-mediated neurodegeneration by affecting the production of a trapped transmembrane isoform of PrP termed CtmPrP (48). Additional studies highlight the unpredictable effects of expressing chimeric PrP constructs on disease outcomes. While Gt mice, referred to as Ki-ChM, expressing a chimeric mouse–human PrP coding sequence containing the mouse PrP signal peptide—as well as mouse PrP sequences at the C terminus—had variable responses to human prions, certain CJD isolates produced disease with mean incubation times in the range of ∼140–180 d (49, 50). When transmitted to Gt mice expressing a different chimeric mouse–human PrP where only the signal peptide is derived from mouse PrP, referred to as Ki-Hu129M/M, mean incubation times of CJD prions that induced disease in Ki-ChM within ∼150 d were prolonged by >300 d (47). With these considerations in mind, and in light of the results reported here, it appears that expression of wild-type human or bovine PrP coding sequences affords the best prospect of conferring susceptibility to CJD and BSE prions in Gt mice.
Cervid PrP Residue 226 Dictates the Propagation of Distinct Strain Conformers. The use of a Gt approach allowed us to unequivocally ascertain the effects of amino acid differences at codon 226 of deer and elk PrP on CWD pathogenesis. Because GtE226+/+ and GtQ226+/+ mice (and for that matter GtE226+/− and GtQ226+/− mice) produce equivalent amounts of elk and deer PrPC, and because they are otherwise syngeneic, the consistently different kinetics of CWD onset can be unambiguously ascribed to the effects of amino acid variation at residue 226. Our findings also demonstrate that Gt mice are susceptible not only to CWD prions, but also to experimentally adapted cervid prions, including cervid-adapted SSBP/1 and cervid-adapted RML, with variable kinetic effects of the residue 226 dimorphism. The production of Gt mice on an FVB background, and the availability of PrP knockout and previously produced Tg mice on the equivalent inbred mouse background, provided the added advantage of allowing us to compare the effects of residue 226 over a range of transgene expression in a controlled genetic context. The consistently more rapid time to clinical disease in mice expressing elk PrPC compared with their Gt and Tg counterparts expressing deer PrPC across a spectrum of transgene expression emphasizes the impact of residue 226 on CWD. Indeed, the comparative analyses of Tg and Gt mice reported here confirms that the variable responses of Tg5037+/− and Tg1536+/− mice to CWD prions originally reported in limited transmission studies (25), resulted from effects of amino acid differences at residue 226 rather than uncontrolled artifacts of transgene expression.
Our findings are in accordance with the notion that residue 226 in elk and deer PrPC acts to select and propagate distinct strains from an ensemble of PrPSc conformers by a mechanism that is compatible with the conformational selection model (19). In support of this view, GtE226+/+ and GtQ226+/+ mice have distinct times to disease onset and neuropathological outcomes in response to intracerebral challenge with the same CWD prion inocula, and the resulting E226 and Q226 prions produced in Gt mice have distinct conformational properties. These findings raise the possibility that the strain properties of CWD prions produced in diseased elk are distinct from those of prions causing CWD in deer. Limited analyses showing distinct pathogenic outcomes between CWD-affected deer and elk (51, 52) are consistent with this interpretation. In light of the influence of strain properties on interspecies prion transmission, our findings that elk and deer PrPC select and propagate different strains are relevant when considering the risk of interspecies CWD transmission, in particular when considering the barrier controlling human susceptibility.
Of note, the effects of amino acid differences at residue 226 on the biological and biochemical properties of CWD prions reported here are supported by structural investigations showing that the distal region of α-helix 3, which contains residue 226, participates with residues in the β2–α2 loop to form a solvent-accessible discontinuous epitope (53, 54). Our subsequent molecular dynamics analyses (55) indicated that E and Q variations at residue 226, as well as the protective polymorphism at the adjacent residue 225 in deer PrP, affected hydrogen bonding between key residues in the β2–α2 loop that modify the strength of interaction between these subdomains, supporting the notion that the plasticity of this discontinuous epitope modulates the efficiency of PrPC to PrPSc conversion.
Investigations of Peripheral CWD Pathogenesis and Contagious Transmission in Gt Mice. Replication of CWD prions to high titers in the peripheral lymphoid system and their resultant shedding in excrement and bodily fluids of diseased cervids are considered to be the underlying features contributing to the unparalleled transmission efficiency of CWD (5). Because they are purported to express PrP at physiological levels in all appropriate cell types, Gt mice have been endorsed as ideal models in which to study peripheral pathogenesis of prion diseases (38). Our findings confirm that this is the case. Gt mice offer a refined improvement on previously described Tg models of CWD, and a new application to explore peripheral CWD pathogenesis and contagious spread of CWD prions. Remarkably, GtQ226+/+ mice showed no difference in time to disease onset following intraperitoneal or intracerebral challenges with CWD (Fig. 4B). In contrast, despite robust PrP over expression in the CNS of Tg1536+/− mice (24), which resulted in more rapid times to disease following intracerebral challenge than counterpart GtQ226+/+(ic) mice expressing physiological levels of PrP, the response of Tg1536+/− mice to intraperitoneal challenge with CWD prions was highly inefficient, reflected in prolonged times to disease onset compared with GtQ226+/+(ip) mice (Fig. 4C). We interpret this to mean that the cos.SHaTet cosmid used to create Tg1536+/+ mice (24) failed to reconstitute appropriate PrP expression in all compartments required for effective peripheral pathogenesis. In accordance with this interpretation, while spleens of diseased GtQ226+/+ mice accumulated PrPSc following either intracerebral or intraperitoneal inoculation, PrPSc was undetectable by Western blotting in spleens of diseased Tg1536+/+ mice that were challenged by the intracerebral or intraperitoneal route (Fig. 4G).
We also show that the route of CWD prion inoculation elicited a pronounced effect on the distribution of cervid PrPSc-containing lesions in the CNS of Gt mice. Accordingly, while intracerebral inoculation of GtE226+/+ mice with 99W12389 CWD prions resulted in widespread, diffuse, and bilaterally symmetrical PrPSc deposition in cortical, hippocampal, thalamic, and hypothalamic regions (Fig. 3), the pattern of E226-cervid PrPSc deposition in GtE226+/+ mice challenged intraperitoneally with the same CWD prions was different from, and more restricted than in GtE226+/+(ic) mice (Fig. 5). Interestingly, when prions from the brains of these mice were serially passaged to additional GtE226+/+ mice by intracerebral inoculation, the pattern of cervid PrPSc deposition in these GtE226+/+(ip-ic) mice reverted to that seen in GtE226+/+ mice intracerebrally challenged with 99W12389 (Fig. 5G). These findings are consistent with the results of previous findings in wild-type mice infected with experimentally adapted scrapie prions, which showed that route of administration affected the profile of neuropathological lesions (56), and underscore the importance of inoculation route when defining prion strain properties. In light of their highly efficient responses to peripherally administered prions, we reasoned that Gt mice might provide an effective means to model horizontal CWD transmission. Our preliminary findings show Gt mice to be a tractable experimental model in which to ascertain the underlying basis for the remarkably contagious transmission of CWD prions (46).
In conclusion, the Gt mice described here represent an important resource with which to address multiple unresolved issues relating to CWD. Our findings describing the role of amino acid differences at residue 226 of deer and elk PrP in dictating the species-specific selection and propagation of cervid prion strains has important implications for assessing the zoonotic potential of established, as well as newly emergent forms of CWD. Of immediate significance, the origins of CWD in Europe (10, 11), and for that matter North American CWD, are enigmatic, and the relationship, if any, between CWD prions from these locations is currently unclear. Understanding whether residue 226 plays a role in the selection and propagation of novel emergent strains in this context would appear to be of particular importance. While prions causing CWD in South Korea are considered to be derivative of North American CWD (7, 8), whether the properties of these CWD prions were altered as a result of passage through indigenous cervid species also remains to be determined. Comparing the properties of North American, European, and Asian CWD prions using Gt mice represents an important first step to assess the prospect of novel global CWD epidemics with unpredictable ecological and public health consequences. Finally, the refined strategies for PrP gene replacement reported here suggest opportunities for improved Gt mouse models in which to study aspects of human and animal prion biology that have until now not been possible using conventional Tg approaches. The generation of optimized Gt mice expressing human PrP rather than chimeric mouse–human PrP constructs would appear to be of particular importance in this regard.
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PRION 2019 ABSTRACTS
1. Interspecies transmission of the chronic wasting disease agent
Justin Greenlee
Virus and Prion Research Unit, National Animal Disease Center, USDA Agriculture Research Service
ABSTRACT
The presentation will summarize the results of various studies conducted at our research center that assess the transmissibility of the chronic wasting disease (CWD) agent to cattle, pigs, raccoons, goats, and sheep. This will include specifics of the relative attack rates, clinical signs, and microscopic lesions with emphasis on how to differentiate cross-species transmission of the CWD agent from the prion diseases that naturally occur in hosts such as cattle or sheep. Briefly, the relative difficulty of transmitting the CWD agent to sheep and goats will be contrasted with the relative ease of transmitting the scrapie agent to white-tailed deer.
53. Evaluation of the inter-species transmission potential of different CWD isolates
Rodrigo Moralesa, Carlos Kramma,b, Paulina Sotoa, Adam Lyona, Sandra Pritzkowa, Claudio Sotoa
aMitchell Center for Alzheimer’s disease and Related Brain Disorders, Dept. of Neurology, McGovern School of Medicine University of Texas Health Science Center at Houston, TX, USA; bFacultad de Medicina, Universidad de los Andes, Santiago, Chile
ABSTRACT
Chronic Wasting Disease (CWD) has reached epidemic proportions in North America and has been identified in South Korea and Northern Europe. CWD-susceptible cervid species are known to share habitats with humans and other animals entering the human food chain. At present, the potential of CWD to infect humans and other animal species is not completely clear. The exploration of this issue acquires further complexity considering the differences in the prion protein sequence due to species-specific variations and polymorphic changes within species. While several species of cervids are naturally affected by CWD, white-tailed deer (WTD) is perhaps the most relevant due to its extensive use in hunting and as a source of food. Evaluation of inter-species prion infections using animals or mouse models is costly and time consuming. We and others have shown that the Protein Misfolding Cyclic Amplification (PMCA) technology reproduces, in an accelerated and inexpensive manner, the inter-species transmission of prions while preserving the strain features of the input PrPSc. In this work, we tested the potential of different WTD-derived CWD isolates to transmit to humans and other animal species relevant for human consumption using PMCA. For these experiments, CWD isolates homozygous for the most common WTD-PrP polymorphic changes (G96S) were used (96SS variant obtained from a pre-symptomatic prion infected WTD). Briefly, 96GG and 96SS CWD prions were adapted in homologous or heterologous substrate by PMCA through several (15) rounds. End products, as well as intermediates across the process, were tested for their inter-species transmission potentials. A similar process was followed to assess seed-templated misfolding of ovine, porcine, and bovine PrPC. Our results show differences on the inter-species transmission potentials of the four adapted materials generated (PrPC/PrPSc polymorphic combinations), being the homologous combinations of seed/substrate the ones with the greater apparent zoonotic potential. Surprisingly, 96SS prions adapted in homologous substrate were the ones showing the easiest potential to template PrPC misfolding from other animal species. In summary, our results show that a plethora of different CWD isolates, each comprising different potentials for inter-species transmission, may exist in the environment. These experiments may help to clarify an uncertain and potentially worrisome public health issue. Additional research in this area may be useful to advise on the design of regulations intended to stop the spread of CWD and predict unwanted zoonotic events.
56. Understanding chronic wasting disease spread potential for at-risk species
Catherine I. Cullingham, Anh Dao, Debbie McKenzie and David W. Coltman
Department of Biological Sciences, University of Alberta, Edmonton AB, Canada
CONTACT Catherine I. Cullingham cathy.cullingham@ualberta.ca
ABSTRACT
Genetic variation can be linked to susceptibility or resistance to a disease, and this information can help to better understand spread-risk in a population. Wildlife disease incidence is increasing, and this is resulting in negative impacts on the economy, biodiversity, and in some instances, human health. If we can find genetic variation that helps to inform which individuals are susceptible, then we can use this information on at-risk populations to better manage negative consequences. Chronic wasting disease, a fatal, transmissible spongiform encephalopathy of cervids (both wild and captive), continues to spread geographically, which has resulted in an increasing host-range. The disease agent (PrPCWD) is a misfolded conformer of native cellular protein (PrPC). In Canada, the disease is endemic in Alberta and Saskatchewan, infecting primarily mule deer and white-tail deer, with a smaller impact on elk and moose populations. As the extent of the endemic area continues to expand, additional species will be exposed to this disease, including bison, bighorn sheep, mountain goat, and pronghorn antelope. To better understand the potential spread-risk among these species, we reviewed the current literature on species that have been orally exposed to CWD to identify susceptible and resistant species. We then compared the amino acid polymorphisms of PrPC among these species to determine whether any sites were linked to susceptibility or resistance to CWD infection. We sequenced the entire PrP coding region in 578 individuals across at-risk populations to evaluate their potential susceptibility. Three amino acid sites (97, 170, and 174; human numbering) were significantly associated with susceptibility, but these were not fully discriminating. All but one species among the resistant group shared the same haplotype, and the same for the susceptible species. For the at-risk species, bison had the resistant haplotype, while bighorn sheep and mountain goats were closely associated with the resistant type. Pronghorn antelope and a newly identified haplotype in moose differed from the susceptible haplotype, but were still closely associated with it. These data suggest pronghorn antelope will be susceptible to CWD while bison are likely to be resistant. Based on this data, recommendations can be made regarding species to be monitored for possible CWD infection.
KEYWORDS: Chronic wasting disease; Prnp; wildlife disease; population genetics; ungulates
75. Mortality surveillance of individuals potentially exposed to chronic wasting disease
Ryan A. Maddoxa, Rachel F. Klosb, Suzanne N. Gibbons-Burgenerb, Bobbi L. Bryanta, Joseph Y. Abramsa, Brian S. Applebyc, Lawrence B. Schonbergera and Ermias D. Belaya
aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA; bWisconsin Department of Health Services, Division of Public Health, Madison, WI, USA; cNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, OH, USA
CONTACT Ryan A. Maddox rmaddox@cdc.gov
ABSTRACT
Introduction: Chronic wasting disease (CWD) is a prion disease of cervids. It is unknown whether CWD prions can infect people; if so, transmission would most likely occur through consumption of meat from infected animals. Since 2003, Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel have maintained a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from cervids positive for CWD. This data source makes it possible to evaluate causes of mortality in individuals potentially exposed to CWD.
Methods: The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e. those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with the National Prion Disease Pathology Surveillance Center (NPDPSC) neuropathology database. Vital status of individuals with date of birth available will be tracked through the identification of possible matches in the National Death Index (NDI) and the evaluation of corresponding cause of death codes.
Results: The database consists of 1561 records for hunt years 2003–2017. Of these, 613 records had accompanying date of birth; 14 entries were removed as duplicates, 1 of whom had consumed venison from a CWD-positive deer during three different hunt years, leaving 599 unique individuals for pending submission to NDI. Of these individuals, 265 of 399 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among persons in the database cross-checked with NPDPSC data.
Conclusion: Because of the robust data link between person and CWD-positive animal, reviewing the cause of mortality in potentially exposed persons is possible; those individuals who self-processed and consumed the meat are likely the best source of information about the potential for zoonotic transmission. The expected long incubation period, should transmission to humans occur, necessitates many years of vital status tracking.
77. Assessing chronic wasting disease strain differences in free-ranging cervids across the United States
Kaitlyn M. Wagnera, Caitlin Ott-Connb, Kelly Strakab, Bob Dittmarc, Jasmine Battend, Robyn Piercea, Mercedes Hennessya, Elizabeth Gordona, Brett Israela, Jenn Ballarde and Mark D Zabela
aPrion Research Center at Colorado State University; bMichigan Department of Natural Resources; cTexas Parks and Wildlife Department; dMissouri Department of Conservation, 5. Arkansas Game and Fish Commission
CONTACT Kaitlyn M. Wagner miedkait@rams.colostate.edu
ABSTRACT
Background/Introduction: Chronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk, and reindeer. Since the initial description of the disease in the 1960’s, CWD has spread to 23 states, 3 Canadian Provinces, South Korea, Norway and, most recently, Finland. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and has not been characterized. Previous studies have shown that there are two distinct strains of CWD. However, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity.
Materials and Methods: To address these knowledge gaps, we used biochemical tests to assess strain differences between CWD outbreaks in Michigan, Texas, Missouri, and Colorado, USA. Brain or lymph node samples were homogenized and digested in 50 µg/mL proteinase K (PK). These samples were then run on a Western blot to assess glycoform ratio and electrophoretic mobility. Texas samples were digested in 100 µg/mL PK. To assess conformational stability, brain or lymph node homogenates were incubated in increasing concentrations of guanidine hydrochloride from 0 M to 4 M in 0.5 M increments. Samples were then precipitated in methanol overnight, washed and PK digested in 50 µg/mL PK before slot blotting.
Results: Our results have found significant differences in glycoform ratio between CWD from Michigan and Colorado, but no differences were observed in conformational stability assays. Interestingly, when testing our CWD isolates from Texas to analyse electrophoretic mobility and glycoform ratio, we found that these samples did not exhibit the characteristic band shift when treated with PK, but PK resistant material remained. Additionally, results from our conformational stability assay demonstrate a unique profile of these Texas isolates. Testing of samples from Missouri is currently underway.
Conclusions: Thus far, our data indicate that there are strain differences between CWD circulating in Michigan and CWD in Colorado and provide important insight into CWD strain differences between two non-contiguous outbreaks. We have also identified a unique strain of CWD in Texas with biochemical strain properties not seen in any of our other CWD isolates. These results highlight the importance of continued surveillance to better understand this devastating disease. These results have important implications for CWD emergence, evolution and our understanding of prion strain heterogeneity on the landscape.
115. Diversity of chronic wasting disease prion strains
Camilo Duque Velásqueza, Elizabeth Triscotta, Chiye Kima, Jacques Van der Merwea, Samia Hannaouib, Trent Bollingerc, Christina Carlsond, Sylvie Benestade, Sabine Gilchb, Judd Aikenf and Debbie McKenziea
aDepartment of Biological Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada; bDepartment of Ecosystem and Public Health, Calgary Prion Research Unit, University of Calgary, Calgary, Canada; cDepartment of Veterinary Pathology, Canadian Wildlife Health Cooperative, University of Saskatchewan, Saskatoon, Canada; dU.S. Geological Survey-National Wildlife Health Center, Madison, WI, USA; eNorwegian Veterinary Institute, Oslo and Trondheim, Norway; fDepartment of Agricultural, Food and Nutritional Sciences, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada
ABSTRACT
Chronic Wasting Disease (CWD) prions affect a variety of cervid species. The expansion of the CWD geographic range and the increasing prevalence makes CWD a concern for wildlife, livestock, and human health. Prion pathogenesis results from the template-directed misfolding of cellular prion proteins (PrPC) into conformational species (e.g. PrPCWD) associated with distinct strains. We propose that cervid cellular prion protein (PrPC) polymorphisms have resulted in prion conformational diversification and speciation (i.e. by adaptive radiation) resulting in emergence of novel CWD strains. New prion strain conformers emerge following transmission between cervids expressing different PrPC amino acid polymorphisms [1-4]. Emergent CWD strains can have novel transmission properties that enable them to infect host species previously considered resistant, suggesting an increase in zoonotic risk as strain-conformers diversify and evolve [2, 3]. We are comparing field CWD isolates of different cervid species from various regions of North America and Norway and have identified differences in biochemical properties of PrPCWD, in vitro and ex vivo propagation and transmission into transgenic mice expressing deer and elk PrP (tgDeer – 96G, tgDeer – 96S, and tgElk-E226) as well as C57Bl6 mice and hamsters. Variations in PrP-res type and protease sensitivity were observed following treatment with proteinase K. Comparison of the PMCA seeding activity using deer and elk PrPC as substrates revealed differences between elk CWD isolates. ElK21 cells also responded differently to various cervid prions. Transmission was efficient for all isolates in tg33 and tgElk mice; however, only a few isolates were able to propagate in host expressing S96 deer PrPC, which has been shown to impose a strong transmission barrier, providing a means of differential selection of CWD strains [2]. Transmission differences were observed following interspecies transmission. While some white-tailed deer and mule deer isolates failed to transmit into hamsters, other isolates transmitted with a low attack rate but considerable sub-clinical infection. These isolates had a migration pattern similar to the hamster-passaged Wisc-1 strain. Two other isolates, transmitted more efficiently into hamsters and produced two different PrP-res migration profiles compared to Wisc-1-like PrP-res. Transmission of Norwegian moose and reindeer CWD isolates is ongoing; preliminary results will be presented. Our data indicate the existence of at least five different CWD strains based on transmission properties.
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156. Screening and characterization of unusual sCJD cases in a CWD endemic state in the USA
Yihui Liua, Manuel Camachoa, Wenquan Zoua,b,c, Qingzhong Konga,b,c
aDepartment of Pathology, Case Western Reserve University (CWRU), Cleveland, USA; bDepartment of Neurology, CWRU, Cleveland, OH, USA; cNational Center for Regenerative Medicine, CWRU, Cleveland, USA
CONTACT Qingzhong Kong qxk2@case.edu
ABSTRACT
Background: Chronic wasting disease (CWD) has spread to 26 states in the USA and three provinces in Canada, and it has been detected recently in Norway and Finland. Potential CWD zoonosis is a serious public health concern. It is unclear whether CWD transmission to humans has already occurred. We aim to start to address this question by examining all available sCJD cases from a CWD endemic state in the USA.
Methods: Frozen brain tissues from all available sCJD cases archived in the National Prion Disease Pathology Surveillance Center from a US state that has been significantly impacted by CWD were sampled at five brain regions. These brain samples were subjected to detailed biochemical analysis to look for unusual patterns, characteristics, and/or distribution of PrPSc in comparison with sCJD samples from states that have not detected CWD. Unusual cases are further scrutinized for their clinical presentations, histopathological features, and history of cervid hunting and venison consumption.
Results and Conclusions: We have found some unusual sCJD cases in this CWD endemic state. We will report our preliminary findings on their features. Currently there is no convincing evidence to support a direct link to CWD for any of these unusual sCJD cases.
Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the position of the National Prion Disease Pathology Surveillance Center.
Thursday, May 23, 2019
Prion 2019 Emerging Concepts CWD, BSE, SCRAPIE, CJD, SCIENTIFIC PROGRAM Schedule and Abstracts
see full Prion 2019 Conference Abstracts
TEXAS CWD TSE PRION STRAIN UNLIKE ANYTHING EVER SEEN
“Wow,” he said. “Unlike anything we've seen before.”
The prions from the Texas deer were a lot harder to destroy than the ones from the Colorado elk. In fact, the guanidine barely damaged them at all. “We’ve never seen that before in any prion strain, which means that it has a completely different structure than we've ever seen before,” says Zabel. And that suggests that it might be a very different kind of chronic wasting disease. The researchers ran the same test on another Texas deer, with the same results.
One day in late February, in their laboratory in Fort Collins, Colorado, Wagner and Zabel compared the prions from the brains of CWD-infected deer in Texas with those of elk in Colorado. They want to know if the proteins were all mangled in the same way, or not. “If they are different, this would suggest that we have different strain properties, which is evidence as we're building our case that we might have multiple strains of CWD circulating in the U.S.,” says Wagner.
Step one is to see if they’re equally easy to destroy using a chemical called guanidine. The shape of a prion dictates everything, including the way it interacts with an animal’s cells and the ease with which chemicals can unfold it.
“Moment of truth,” said Wagner, as she and Zabel huddled around a computer, waiting for results to come through. When they did, Zabel was surprised.
“Wow,” he said. “Unlike anything we've seen before.”
The prions from the Texas deer were a lot harder to destroy than the ones from the Colorado elk. In fact, the guanidine barely damaged them at all. “We’ve never seen that before in any prion strain, which means that it has a completely different structure than we've ever seen before,” says Zabel. And that suggests that it might be a very different kind of chronic wasting disease. The researchers ran the same test on another Texas deer, with the same results.
Now, these are only the preliminary results from a few animals. Wagner and Zabel have a lot more experiments to do. But if future tests come to the same conclusion, it would support their hypothesis that there are multiple strains of chronic wasting disease out there, all with different origins. That, in turn, could mean that this disease will become even trickier to manage than it already is.
And, Zabel adds, there’s something else. “If it's still evolving, it may still evolve into a form that could potentially, eventually affect humans,” he says.
Zabel is not the only one worried about that possibility.
OSTERHOLM, THE EPIDEMIOLOGIST from Minnesota, is also concerned. He directs the Center for Infectious Disease Research and Policy at the University of Minnesota, and is serving a one-year stint as a “Science Envoy for Health Security” with the U.S. State Department. In February, he told Minnesota lawmakers that when it comes to chronic wasting disease, we are playing with fire. “You are going to hear from people that this is not going to be a problem other than a game farm issue. You're going to hear from people that it's not going to transmit to people, and I hope they're right, but I wouldn't bet on it,” he said. “And if we lose this one and haven’t done all we can do, we will pay a price.”
If that wasn’t warning enough, he added: “Just remember what happened in England.”
SUNDAY, APRIL 14, 2019
Chronic Wasting Disease TSE Prion Strains everything in Texas is bigger, better, and badder
WEDNESDAY, MAY 15, 2019
TAHC CWD TSE Prion Summary Minutes of the 402nd Commission Meeting – 12/11/2018
FRIDAY, APRIL 05, 2019
TPWD CWD Sampling Effort Leads to Proposed Containment Zone Expansion
TUESDAY, MARCH 05, 2019
TAHC CWD TSE PRION AT 144 POSITIVE MINUTES OF THE 401st COMMISSION MEETING Texas Animal Health Commission August 7, 2018
TEXAS BREEDER DEER ESCAPEE WITH CWD IN THE WILD, or so the genetics would show?
apparently, no ID though. tell me it ain't so please...
23:00 minute mark
''Free Ranging Deer, Dr. Deyoung looked at Genetics of this free ranging deer and what he found was, that the genetics on this deer were more similar to captive deer, than the free ranging population, but he did not see a significant connection to any one captive facility that he analyzed, so we believe, Ahhhhhh, this animal had some captive ahhh, whatnot.''
SATURDAY, JANUARY 19, 2019
Texas Chronic Wasting Disease CWD TSE Prion Symposium 2018 posted January 2019 VIDEO SET 18 CLIPS
Chronic Wasting Disease CWD TSE Prion aka mad deer disease zoonosis
We hypothesize that:
(1) The classic CWD prion strain can infect humans at low levels in the brain and peripheral lymphoid tissues;
(2) The cervid-to-human transmission barrier is dependent on the cervid prion strain and influenced by the host (human) prion protein (PrP) primary sequence;
(3) Reliable essays can be established to detect CWD infection in humans; and
(4) CWD transmission to humans has already occurred. We will test these hypotheses in 4 Aims using transgenic (Tg) mouse models and complementary in vitro approaches.
(1) The classic CWD prion strain can infect humans at low levels in the brain and peripheral lymphoid tissues;
(2) The cervid-to-human transmission barrier is dependent on the cervid prion strain and influenced by the host (human) prion protein (PrP) primary sequence;
(3) Reliable essays can be established to detect CWD infection in humans; and
(4) CWD transmission to humans has already occurred. We will test these hypotheses in 4 Aims using transgenic (Tg) mouse models and complementary in vitro approaches.
ZOONOTIC CHRONIC WASTING DISEASE CWD TSE PRION UPDATE
here is the latest;
PRION 2018 CONFERENCE
Oral transmission of CWD into Cynomolgus macaques: signs of atypical disease, prion conversion and infectivity in macaques and bio-assayed transgenic mice
Hermann M. Schatzl, Samia Hannaoui, Yo-Ching Cheng, Sabine Gilch (Calgary Prion Research Unit, University of Calgary, Calgary, Canada) Michael Beekes (RKI Berlin), Walter Schulz-Schaeffer (University of Homburg/Saar, Germany), Christiane Stahl-Hennig (German Primate Center) & Stefanie Czub (CFIA Lethbridge).
To date, BSE is the only example of interspecies transmission of an animal prion disease into humans. The potential zoonotic transmission of CWD is an alarming issue and was addressed by many groups using a variety of in vitro and in vivo experimental systems. Evidence from these studies indicated a substantial, if not absolute, species barrier, aligning with the absence of epidemiological evidence suggesting transmission into humans. Studies in non-human primates were not conclusive so far, with oral transmission into new-world monkeys and no transmission into old-world monkeys. Our consortium has challenged 18 Cynomolgus macaques with characterized CWD material, focusing on oral transmission with muscle tissue. Some macaques have orally received a total of 5 kg of muscle material over a period of 2 years.
After 5-7 years of incubation time some animals showed clinical symptoms indicative of prion disease, and prion neuropathology and PrPSc deposition were detected in spinal cord and brain of some euthanized animals. PrPSc in immunoblot was weakly detected in some spinal cord materials and various tissues tested positive in RT-QuIC, including lymph node and spleen homogenates. To prove prion infectivity in the macaque tissues, we have intracerebrally inoculated 2 lines of transgenic mice, expressing either elk or human PrP. At least 3 TgElk mice, receiving tissues from 2 different macaques, showed clinical signs of a progressive prion disease and brains were positive in immunoblot and RT-QuIC. Tissues (brain, spinal cord and spleen) from these and pre-clinical mice are currently tested using various read-outs and by second passage in mice. Transgenic mice expressing human PrP were so far negative for clear clinical prion disease (some mice >300 days p.i.). In parallel, the same macaque materials are inoculated into bank voles.
Taken together, there is strong evidence of transmissibility of CWD orally into macaques and from macaque tissues into transgenic mouse models, although with an incomplete attack rate.
The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology.
Our ongoing studies will show whether the transmission of CWD into macaques and passage in transgenic mice represents a form of non-adaptive prion amplification, and whether macaque-adapted prions have the potential to infect mice expressing human PrP.
The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD..
***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <***
https://prion2018.org/
READING OVER THE PRION 2018 ABSTRACT BOOK, LOOKS LIKE THEY FOUND THAT from this study ;
P190 Human prion disease mortality rates by occurrence of chronic wasting disease in freeranging cervids, United States
Abrams JY (1), Maddox RA (1), Schonberger LB (1), Person MK (1), Appleby BS (2), Belay ED (1) (1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA..
SEEMS THAT THEY FOUND Highly endemic states had a higher rate of prion disease mortality compared to non-CWD
states.
AND ANOTHER STUDY;
P172 Peripheral Neuropathy in Patients with Prion Disease
Wang H(1), Cohen M(1), Appleby BS(1,2) (1) University Hospitals Cleveland Medical Center, Cleveland, Ohio (2) National Prion Disease Pathology Surveillance Center, Cleveland, Ohio..
IN THIS STUDY, THERE WERE autopsy-proven prion cases from the National Prion Disease Pathology Surveillance Center that were diagnosed between September 2016 to March 2017,
AND
included 104 patients. SEEMS THEY FOUND THAT The most common sCJD subtype was MV1-2 (30%), followed by MM1-2 (20%),
AND
THAT The Majority of cases were male (60%), AND half of them had exposure to wild game.
snip...
see more on Prion 2017 Macaque study from Prion 2017 Conference and other updated science on cwd tse prion zoonosis below...terry
https://prion2018.org/wp-content/uploads/2018/05/program.pdf
https://prion2018.org/
Thursday, May 23, 2019
Prion 2019 Emerging Concepts CWD, BSE, SCRAPIE, CJD, SCIENTIFIC PROGRAM Schedule and Abstracts
see full Prion 2019 Conference Abstracts
FRIDAY, MAY 24, 2019
Assessing chronic wasting disease strain differences in free-ranging cervids across the United States
MONDAY, MAY 20, 2019
APHIS, USDA, Announces the Finalized Chronic Wasting Disease Herd Certification Program Standards Singeltary Submissions
WEDNESDAY, APRIL 03, 2019
Estimating the amount of Chronic Wasting Disease infectivity passing through abattoirs and field slaughter
WEDNESDAY, MARCH 13, 2019
CWD, TSE, PRION, MATERNAL mother to offspring, testes, epididymis, seminal fluid, and blood
THURSDAY, MARCH 14, 2019
USDA APHIS CDC Cervids: Chronic Wasting Disease Specifics Updated 2019
SATURDAY, MARCH 16, 2019
Chronic Wasting Disease CWD TSE Prion United States of America Update March 16, 2019
TUESDAY, MARCH 26, 2019
USDA ARS 2018 USAHA RESOLUTIONS Investigation of the Role of the Prion Protein Gene in CWD Resistance and Transmission of Disease
TUESDAY, APRIL 30, 2019
Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies 2018 Annual Report
THURSDAY, OCTOBER 04, 2018
Cervid to human prion transmission 5R01NS088604-04 Update
MONDAY, APRIL 01, 2019
PUBLIC HEALTH U of M launches Chronic Wasting Disease Program to address potential health crisis
SATURDAY, FEBRUARY 23, 2019
Chronic Wasting Disease CWD TSE Prion and THE FEAST 2003 CDC an updated review of the science 2019
TUESDAY, NOVEMBER 04, 2014
Six-year follow-up of a point-source exposure to CWD contaminated venison in an Upstate New York community: risk behaviours and health outcomes 2005–2011
Authors, though, acknowledged the study was limited in geography and sample size and so it couldn't draw a conclusion about the risk to humans. They recommended more study. Dr. Ermias Belay was the report's principal author but he said New York and Oneida County officials are following the proper course by not launching a study. "There's really nothing to monitor presently. No one's sick," Belay said, noting the disease's incubation period in deer and elk is measured in years. "
MONDAY, FEBRUARY 25, 2019
***> MAD DOGS AND ENGLISHMEN BSE, SCRAPIE, CWD, CJD, TSE PRION A REVIEW 2019
Scientific Advisors and Consultants Staff 2001 Advisory Committee TSE PRION Singeltary Submission Freas Monday, January 08,2001 3:03 PM FDA Singeltary submission 2001
Greetings again Dr. Freas and Committee Members,
I wish to submit the following information to the Scientific Advisors and Consultants Staff 2001 Advisory Committee (short version). I understand the reason of having to shorten my submission, but only hope that you add it to a copy of the long version, for members to take and read at their pleasure, (if cost is problem, bill me, address below). So when they realize some time in the near future of the 'real' risks i speak of from human/animal TSEs and blood/surgical products. I cannot explain the 'real' risk of this in 5 or 10 minutes at some meeting, or on 2 or 3 pages, but will attempt here:
fda link is dead in the water;
snip...see full text
CHRONIC WASTING DISEASE CONGRESS Serial No. 107-117 May 16, 2002
CHRONIC WASTING DISEASE
JOINT OVERSIGHT HEARING BEFORE THE SUBCOMMITTEE ON FORESTS AND FOREST HEALTH JOINT WITH THE SUBCOMMITTEE ON FISHERIES CONSERVATION, WILDLIFE AND OCEANS OF THE COMMITTEE ON RESOURCES U.S. HOUSE OF REPRESENTATIVES ONE HUNDRED SEVENTH CONGRESS SECOND SESSION
May 16, 2002
Serial No. 107-117
snip...
Mr. MCINNIS. Today, this joint Subcommittee hearing will explore an issue of immeasurable importance to the growing number of communities in wide-ranging parts of this country, the growing incidence of Chronic Wasting Disease in North America’s wild and captive deer and elk populations. In a matter of just a few months, this once parochial concern has grown into something much larger and much more insidious than anyone could have imagined or predicted.
As each day passes, this problem grows in its size, scope, and consequence. One thing becomes clear. Chronic Wasting Disease is not a Colorado problem. It is a Wisconsin problem or a Nebraska or Wyoming problem. It is a national problem and anything short of a fully integrated, systematic national assault on this simply will not do, which is precisely why we brought our group together here today.
snip...
So this is a disease that is spreading throughout the continent and it is going to require a national response as well as the efforts that are currently taking place in States like Wisconsin, Colorado, Nebraska, Wyoming, the interest they now have down in Texas and some of the neighboring States that have large white-tailed deer population and also elk.
This is a huge issue for us, Mr. Chairman, in the State of Wisconsin. I want to commend Governor McCallum and your staff and the various agencies for the rapid response that you have shown, given the early detection of CWD after the last deer hunting season. The problem that we have, though, is just a lack of information, good science in regards to what is the best response, how dangerous is this disease. We cannot close the door, quite frankly, with the paucity of scientific research that is out there right now in regards to how the disease spreads, the exposure of other livestock herds—given the importance of our dairy industry in the State, that is a big issue—and also the human health effects.
the British disease...NOT, the UKBSEnvCJD only theory was/is bogus $$$
*** USA sporadic CJD MAD COW DISEASE HAS HUGE PROBLEM Video
*** sporadic CJD linked to mad cow disease
*** you can see video here and interview with Jeff's Mom, and scientist telling you to test everything and potential risk factors for humans ***
WEDNESDAY, MAY 29, 2019
The European Union Summary Report On Surveillance For The Presence Of Transmissible Spongiform Encephalopathies (TSE): The Situation In 2017
friendly fire, pass it forward, they call it iatrogenic cjd, or what i call 'tse prion poker', are you all in $$$
SATURDAY, MARCH 16, 2019
Medical Devices Containing Materials Derived from Animal Sources (Except for In Vitro Diagnostic Devices) Guidance for Industry and Food and Drug Administration Staff Document issued on March 15, 2019 Singeltary Submission
TUESDAY, APRIL 09, 2019
Horizon Health Network Moncton Hospital notified more than 700 patients after two cases of CJD were diagnosed both patients had undergone cataracts surgery before being diagnosed
TUESDAY, NOVEMBER 20, 2018
CDC Eyes of CJD patients show evidence of prions concerns for iatrogenic transmission
WEDNESDAY, OCTOBER 17, 2018
PRICE OF TSE PRION POKER GOES UP spectrum of human prion diseases may extend the current field and may notably include spinal cord diseases
REVIEW
***> In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. <***
***> The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology.<***
***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <***
***> All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals.<***
***> In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids.'' Scientific opinion on chronic wasting disease (II) <***
Saturday, February 2, 2019
CWD GSS TSE PRION SPINAL CORD, Confucius Ponders, What if?
SUNDAY, MARCH 10, 2019
National Prion Disease Pathology Surveillance Center Cases Examined¹ Updated Feb 1, 2019 Variably protease-sensitive prionopathy VPSPr
Subject: Prion 2019 Conference
Thursday, May 23, 2019
Prion 2019 Emerging Concepts CWD, BSE, SCRAPIE, CJD, SCIENTIFIC PROGRAM Schedule and Abstracts
see full Prion 2019 Conference Abstracts
FRIDAY, MAY 24, 2019
Assessing chronic wasting disease strain differences in free-ranging cervids across the United States
MONDAY, MAY 20, 2019
APHIS, USDA, Announces the Finalized Chronic Wasting Disease Herd Certification Program Standards Singeltary Submissions
terry
posted by Terry S. Singeltary Sr. at
11:34 AM
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