Chronic Wasting Disease Pathogenesis Research Articles

Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

Gene-Edited Cell Models to Study Chronic Wasting Disease

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrPC), PrPSc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies.

Large animal models for chronic wasting disease.

Chronic wasting disease (CWD) is a fatal neurodegenerative prion disease of cervid species including deer, elk, moose and reindeer. The disease has shown both geographic and species expansion since its discovery in the late 1960’s and is now recognized in captive and free-ranging cervid populations in North America, Asia and Europe. The facile transmission of CWD is unique among prion diseases and has resulted in growing concern for cervid populations and human public health. The development of native cervid host models with longitudinal monitoring has revealed new insights about CWD pathogenesis and transmission dynamics. More than 20 years of experimental studies conducted in these models, using biologically relevant routes of infection, have led to better understanding of many aspect of CWD infections. This review addresses some of these insights, including: (i) the temporal intra-host trafficking of CWD prions in tissues and bodily fluids, (ii) the presence of infectivity shed in bodily excretions that may help explain the facile transmission of CWD, (iii) mother-to-offspring CWD transmission, (iv) the influence of some Prnp polymorphisms on CWD susceptibility, and (vi) continued development of vaccine strategies to mitigate CWD.

Neural transcriptomic signature of chronic wasting disease in white-tailed deer

BackgroundThe increasing prevalence and expanding geographical range of the chronic wasting disease (CWD) panzootic in cervids is threatening human, animal, environmental and economic health. The pathogenesis of CWD in cervids is, however, not well understood. We used RNA sequencing (RNA-seq) to compare the brain transcriptome from white-tailed deer (WTD; Odocoileus virginianus) clinically affected with CWD (n = 3) to WTD that tested negative (n = 8) for CWD. In addition, one preclinical CWD+ brain sample was analyzed by RNA-seq.ResultsWe found 255 genes that were significantly deregulated by CWD, 197 of which were upregulated. There was a high degree of overlap in differentially expressed genes (DEGs) identified when using either/both the reference genome assembly of WTD for mapping sequenced reads to or the better characterized genome assembly of a closely related model species, Bos taurus. Quantitative PCR of a subset of the DEGs confirmed the RNA-seq data. Gene ontology term enrichment analysis found a majority of genes involved in immune activation, consistent with the neuroinflammatory pathogenesis of prion diseases. A metagenomic analysis of the RNA-seq data was conducted to look for the presence of spiroplasma and other bacteria in CWD infected deer brain tissue.ConclusionsThe gene expression changes identified highlight the role of innate immunity in prion infection, potential disease associated biomarkers and potential targets for therapeutic agents. An association between CWD and spiroplasma infection was not found.

COMPARISON OF CHRONIC WASTING DISEASE DETECTION METHODS AND PROCEDURES: IMPLICATIONS FOR FREE-RANGING WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) SURVEILLANCE AND MANAGEMENT.

Throughout North America, chronic wasting disease (CWD) has emerged as perhaps the greatest threat to wild cervid populations, including white-tailed deer (WTD; Odocoileus virginianus). White-tailed deer are the most sought-after big game species across North America with populations of various subspecies in nearly all Canadian provinces, the contiguous US, and Mexico. Documented CWD cases have dramatically increased across the WTD range since the mid-1990s, including in Minnesota, US. CWD surveillance in free-ranging WTD and other cervid populations mainly depends upon immunodetection methods such as immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) on medial retropharyngeal lymph nodes and obex. More recent technologies centered on prion protein amplification methods of detection have shown promise as more sensitive and rapid CWD diagnostic tools. Here, we used blinded samples to test the efficacy of real-time quaking-induced conversion (RT-QuIC) in comparison to ELISA for screening tissues collected in 2019 from WTD in southeastern Minnesota, where CWD has been routinely detected since 2016. Our results support previous findings that RT-QuIC is a more sensitive tool for CWD detection than current antibody-based methods. Additionally, a CWD testing protocol that includes multiple lymphoid tissues (e.g., medial retropharyngeal lymph node, parotid lymph node, and palatine tonsil) per animal can effectively identify a greater number of CWD detections in a WTD population than a single sample type (e.g., medial retropharyngeal lymph nodes). These results show that the variability of CWD pathogenesis, sampling protocol, and testing platform must be considered for the effective detection and management of CWD throughout North America.

Alterations in gut microbiota linked to provenance, sex, and chronic wasting disease in white-tailed deer (Odocoileus virginianus)

Chronic wasting disease (CWD) is a fatal, contagious, neurodegenerative prion disease affecting both free-ranging and captive cervid species. CWD is spread via direct or indirect contact or oral ingestion of prions. In the gastrointestinal tract, prions enter the body through microfold cells (M-cells), and the abundance of these cells can be influenced by the gut microbiota. To explore potential links between the gut microbiota and CWD, we collected fecal samples from farmed and free-ranging white-tailed deer (Odocoileus virginianus) around the Midwest, USA. Farmed deer originated from farms that were depopulated due to CWD. Free-ranging deer were sampled during annual deer harvests. All farmed deer were tested for CWD via ELISA and IHC, and we used 16S rRNA gene sequencing to characterize the gut microbiota. We report significant differences in gut microbiota by provenance (Farm 1, Farm 2, Free-ranging), sex, and CWD status. CWD-positive deer from Farm 1 and 2 had increased abundances of Akkermansia, Lachnospireacea UCG-010, and RF39 taxa. Overall, differences by provenance and sex appear to be driven by diet, while differences by CWD status may be linked to CWD pathogenesis.

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis.

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrPC) to convert into infectious prions (PrPSc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrPC and PrPSc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrPC to PrPSc conversion and determining PrPC stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.

Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice

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.

Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent

BackgroundThe Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD.ResultsElk with longer incubation periods accumulated relatively less PrPSc in the brain than elk with shorter incubation periods. PrPSc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrPSc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrPSc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate.ConclusionsThis study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrPSc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrPSc from MM132 and LM132 elk are similar to each other and different to PrPSc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrPSc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer.

Among prion infections, two scenarios of prion spread are generally observed: (i) early lymphoid tissue replication or (ii) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrPCWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrPCWD was not detected by either method in the initial days (1 and 3) postexposure, we observed PrPCWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months postexposure (MPE). At 3 MPE, PrPCWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrPCWD burden in all lymphoid tissues had increased and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx, and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion.IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids, and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrPCWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state.

Mother to offspring transmission of chronic wasting disease in reeves' muntjac deer.

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.

Detection of Chronic Wasting Disease Prions in Salivary, Urinary, and Intestinal Tissues of Deer: Potential Mechanisms of Prion Shedding and Transmission

Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrP(CWD)-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10(-11)- to 10(0)-fold of that found in brain of the same animal. Deer with highest levels of PrP(CWD) amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrP(CWD) was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall.

Chronic Wasting Disease Prions in Elk Antler Velvet

Chronic wasting disease (CWD) is a contagious, fatal prion disease of deer and elk that continues to emerge in new locations. To explore the means by which prions are transmitted with high efficiency among cervids, we examined prion infectivity in the apical skin layer covering the growing antler (antler velvet) by using CWD-susceptible transgenic mice and protein misfolding cyclic amplification. Our finding of prions in antler velvet of CWD-affected elk suggests that this tissue may play a role in disease transmission among cervids. Humans who consume antler velvet as a nutritional supplement are at risk for exposure to prions. The fact that CWD prion incubation times in transgenic mice expressing elk prion protein are consistently more rapid raises the possibility that residue 226, the sole primary structural difference between deer and elk prion protein, may be a major determinant of CWD pathogenesis.

A prion disease of cervids: Chronic wasting disease

Chronic wasting disease (CWD) is a prion disease of deer, elk, and moose, initially recognized in Colorado mule deer. The discovery of CWD beyond the borders of Colorado and Wyoming, in Canada and as far east as New York, has led to its emergence as a prion disease of international importance. Epidemiological studies indicate that CWD is horizontally transmitted among free-ranging animals, potentially indirectly by prion-containing secreta or excreta contaminating the environment. Experimental CWD transmission attempts to other wild and domestic mammals and to transgenic mice expressing the prion protein of cattle, sheep, and humans have shed light on CWD species barriers. Transgenic mice expressing the cervid prion protein have proven useful for assessing the genetic influences of Prnp polymorphisms on CWD susceptibility. Accumulating evidence of CWD pathogenesis indicates that the misfolded prion protein or prion infectivity seems to be widely disseminated in many nonneural organs and in blood. This review highlights contemporary research findings in this prion disease of free-ranging wildlife.

The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation

The elk prion protein gene (PRNP) encodes either methionine (M) or leucine (L) at codon 132, the L132 allele apparently affording protection against chronic wasting disease (CWD). The corresponding human codon 129 polymorphism influences the host range of bovine spongiform encephalopathy (BSE) prions. To fully address the influence of this cervid polymorphism on CWD pathogenesis, we created transgenic (Tg) mice expressing cervid PrPC with L at residue 132, referred to as CerPrPC-L132, and compared the transmissibility of CWD prions from elk of defined PRNP genotypes, namely homozygous M/M or L/L or heterozygous M/L, in these Tg mice with previously described Tg mice expressing CerPrPC-M132, referred to as Tg(CerPrP) mice. While Tg(CerPrP) mice were consistently susceptible to CWD prions from elk of all three genotypes, Tg(CerPrP-L132) mice uniformly failed to develop disease following challenge with CWD prions. In contrast, SSBP/1 sheep scrapie prions transmitted efficiently to both Tg(CerPrP) and Tg(CerPrP-L132) mice. Our findings suggest that the elk 132 polymorphism controls prion susceptibility at the level of prion strain selection and that cervid PrP L132 severely restricts propagation of CWD prions. We speculate that the L132 polymorphism results in less efficient conversion of CerPrPC-L132 by CWD prions, an effect that is overcome by the SSBP/1 strain. Our studies show the accumulation of subclinical levels of CerPrPSc in aged asymptomatic CWD-inoculated Tg(CerPrP-L132) mice and also suggests the establishment of a latent infection state in apparently healthy elk expressing this seemingly protective allele.

Resistance to Chronic Wasting Disease in Transgenic Mice Expressing a Naturally Occurring Allelic Variant of Deer Prion Protein

Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer or elk PrP in transgenic mice has induced susceptibility to chronic wasting disease (CWD), the prion disease of cervids. In the current experiments, transgenic mice expressing two naturally occurring allelic variants of deer PrP with either glycine (G) or serine (S) at residue 96 were found to differ in susceptibility to CWD infection. G96 mice were highly susceptible to infection, and disease appeared starting as early as 160 days postinfection. In contrast, S96 mice showed no evidence of disease or generation of disease-associated protease-resistant PrP (PrPres) over a 600-day period. At the time of clinical disease, G96 mice showed typical vacuolar pathology and deposition of PrPres in many brain regions, and in some individuals, extensive neuronal loss and apoptosis were noted in the hippocampus and cerebellum. Extraneural accumulation of PrPres was also noted in spleen and intestinal tissue of clinically ill G96 mice. These results demonstrate the importance of deer PrP polymorphisms in susceptibility to CWD infection. Furthermore, this deer PrP transgenic model is the first to demonstrate extraneural accumulation of PrPres in spleen and intestinal tissue and thus may prove useful in studies of CWD pathogenesis and transmission by oral or other natural routes of infection.

Chronic wasting disease in deer and elk: scientific facts and findings.

Chronic wasting disease (CWD) is a prion disease of cervids such as deer and elk in North America. Unlike other transmissible spongiform encephalopathy (TSE) such as scrapie, CWD occurs in both captive and wild ranging animals, but not in domestic ruminants such as sheep and cattle. In this paper, the history of the disease, pathogenesis of CWD, susceptibility of animals, its transmission mechanisms, potential origins of the disease, diagnostic methods in the field and laboratory tests, surveillance and survey systems in the USA and Canada, control strategies, economic impact of the disease, food and feed safety, and the risks in human and animals are reviewed and summarized. Although there is no evidence that CWD has been transmitted to humans, it may have the potential to infect humans.