Chronic Wasting Disease: State of the Science.

Chronic wasting disease (CWD) is a prion disease affecting cervid species, both free-ranging and captive populations. As the geographic range continues to expand and disease prevalence continues to increase, CWD will have an impact on cervid populations, local economies, and ecosystem health. Mitigation of this "wicked" disease will require input from many different stakeholders including hunters, landowners, research biologists, wildlife managers, and others, working together. The NC1209 (North American interdisciplinary chronic wasting disease research consortium) is composed of scientists from different disciplines involved with investigating and managing CWD. Leveraging this broad breadth of expertise, the Consortium has created a state-of-the-science review of five key aspects of CWD, including current diagnostic capabilities for detecting prions, requirements for validating these diagnostics, the role of environmental transmission in CWD dynamics, and potential zoonotic risks associated with CWD. The goal of this review is to increase stakeholders', managers', and decision-makers' understanding of this disease informed by current scientific knowledge.

In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Innate Immune Status of Glia Modulates Prion Propagation in Early Stage of Infection.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion-affected brains is neuroinflammation, histopathologically characterized by reactive gliosis surrounding prion deposition. The cause and effect of these cellular responses are still unclear. Here we investigate the impact of innate immune responses on prion replication using in vitro cell culture models. Hamster-adapted transmissible mink encephalopathy prions, hyper (HY) and drowsy (DY) strains, were assayed for accumulation of pathogenic prion protein (PrPSc) in primary glial cultures derived from 8-day-old hamster pups. The kinetics of PrPSc accumulation largely depended on prion strain and brain regions from where glial cells originated. Glial cells derived from the cerebellum were susceptible to HY, but resistant to DY strain as determined by western blot analysis, immunocytochemistry, and animal bioassay. Glial cells from the cerebral cortex were, however, refractory to both strains. PrPSc accumulation was affected by innate immune modulators. Priming glial cells with lipopolysaccharide decreased prion replication, whereas pre-treatment with dexamethasone, inhibiting innate immunity, increased susceptibility to DY infection. Our results suggest that neuroinflammation resulting from prion infection is a response to resolve and/or prevent prion propagation in the brain. It implies a therapeutic potential of innate immune modulation in the early stages of prion disease.

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging.

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

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.

Movement of Chronic Wasting Disease Prions in Prairie, Boreal and Alpine Soils.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy negatively impacting cervids on three continents. Soil can serve as a reservoir for horizontal transmission of CWD by interaction with the infectious prion protein (PrPCWD) shed by diseased individuals and from infected carcasses. We investigated the pathways for PrPCWD migration in soil profiles using lab-scale soil columns, comparing PrPCWD migration through pure soil minerals (quartz, illite and montmorillonite), and diverse soils from boreal (Luvisol, Brunisol) and prairie (Chernozem) regions. We analyzed the leachate of the soil columns by immunoblot and protein misfolding cyclic amplification (PMCA) and detected PrP in the leachates of columns composed of quartz, illite, Luvisol and Brunisol. Animal bioassay confirmed the presence of CWD infectivity in the leachates from quartz, illite and Luvisol columns. Leachates from columns with montmorillonite and prairie Chernozems did not contain PrP detectable by immunoblotting or PMCA; bioassay confirmed that the Chernozemic leachate was not infectious. Analysis of the solid phase of the columns confirmed the migration of PrP to lower layers in the illite column, while the strongest signal in the montmorillonite column remained close to the surface. Montmorillonite, the prevalent clay mineral in prairie soils, has the strongest prion binding ability; by contrast, illite, the main clay mineral in northern boreal and tundra soils, does not bind prions significantly. This suggests that in soils of North American CWD-endemic regions (Chernozems), PrPCWD would remain on the soil surface due to avid binding to montmorillonite. In boreal Luvisols and mountain Brunisols, prions that pass through the leaf litter will continue to move through the soil mineral horizon, becoming less bioavailable. In light-textured soils where quartz is a dominant mineral, the majority of the infectious prions will move through the soil profile. Local soil properties may consequently determine the efficiency of environmental transmission of CWD.

Sheep scrapie and deer rabies in England prior to 1800.

Eighteenth-century England witnessed the emergence of two neurological diseases in animals. Scrapie, a transmissible spongiform encephalopathy, is a fatal neurodegenerative disease of sheep and goats that appears in classical and atypical forms. Reports of classical scrapie in continental Europe with described symptoms date back to 1750 in what is now western Poland. However, two major outbreaks of scrapie appeared in England prior to the 1800s. References to a sheep disease with a resemblance to scrapie first appear in Southwestern England between 1693 and 1722 and in the East Midlands between 1693 and 1706. Concurrent with the descriptions of scrapie in sheep was a neurological disease of deer first appearing in the East of England. Two 18th-century writers remarked on the symptomatic similarities between the sheep and deer neurological diseases. Multiple outbreaks of the unknown deer disease existing as early as 1772 are examined and are identified as rabies.

Age- and time-dependent mitochondrial genotoxic and myopathic effects of beta-guanidinopropionic acid, a creatine analog, on rodent skeletal muscles.

Beta-guanidinopropionic acid (GPA) is a creatine analog suggested as a treatment for hypertension, diabetes, and obesity, which manifest primarily in older adults. A notable side effect of GPA is the induction of mitochondrial DNA deletion mutations. We hypothesized that mtDNA deletions contribute to muscle aging and used the mutation promoting effect of GPA to examine the impact of mtDNA deletions on muscles with differential vulnerability to aging. Rats were treated with GPA for up to 4 months starting at 14 or 30 months of age. We examined quadriceps and adductor longus muscles as the quadriceps exhibits profound age-induced deterioration, while adductor longus is maintained. GPA decreased body and muscle mass and mtDNA copy number while increasing mtDNA deletion frequency. The interactions between age and GPA treatment observed in the quadriceps were not observed in the adductor longus. GPA had negative mitochondrial effects in as little as 4 weeks. GPA treatment exacerbated mtDNA deletions and muscle aging phenotypes in the quadriceps, an age-sensitive muscle, while the adductor longus was spared. GPA has been proposed for use in age-associated diseases, yet the pharmacodynamics of GPA differ with age and include the detrimental induction of mtDNA deletions, a mitochondrial genotoxic stress that is pronounced in muscles that are most vulnerable to aging. Further research is needed to determine if the proposed benefits of GPA on hypertension, diabetes, and obesity outweigh the detrimental mitochondrial and myopathic side effects.

Emergence of CWD strains.

Chronic wasting disease (CWD) strains present a novel challenge to defining and mitigating this contagious prion disease of deer, elk, moose, and reindeer. Similar to strains of other prion diseases (bovine spongiform encephalopathy, sheep scrapie), CWD strains can affect biochemical and neuropathological properties of the infectious agent, and importantly interspecies transmission. To date, ten CWD strains have been characterized. The expanding range of CWD in North America and its presence in South Korea as well as Scandinavian countries will potentially result in millions of cervids infected with CWD; thus, novel strains will continue to emerge. In this review, we will summarize the characteristics of known CWD strains and describe the impact of prion protein gene polymorphisms on the generation of strains. We will also discuss the evidence that individual cervids can harbor more than one CWD strain, complicating strain analysis, and affecting selection and adaptation of strains in new hosts.

Chronic wasting disease prions in mule deer interdigital glands.

Chronic wasting disease (CWD) is a geographically expanding, fatal neurodegenerative disease in cervids. The disease can be transmitted directly (animal-animal) or indirectly via infectious prions shed into the environment. The precise mechanisms of indirect CWD transmission are unclear but known sources of the infectious prions that contaminate the environment include saliva, urine and feces. We have previously identified PrPC expression in deer interdigital glands, sac-like exocrine structures located between the digits of the hooves. In this study, we assayed for CWD prions within the interdigital glands of CWD infected deer to determine if they could serve as a source of prion shedding and potentially contribute to CWD transmission. Immunohistochemical analysis of interdigital glands from a CWD-infected female mule deer identified disease-associated PrPCWD within clusters of infiltrating leukocytes adjacent to sudoriferous and sebaceous glands, and within the acrosyringeal epidermis of a sudoriferous gland tubule. Proteinase K-resistant PrPCWD material was amplified by serial protein misfolding cyclic amplification (sPMCA) from soil retrieved from between the hoof digits of a clinically affected mule deer. Blinded testing of interdigital glands from 11 mule deer by real-time quake-induced conversion (RT-QuIC) accurately identified CWD-infected animals. The data described suggests that interdigital glands may play a role in the dissemination of CWD prions into the environment, warranting future investigation.

Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats: A short report.

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir's effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD.

Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.

Susceptibility of Beavers to Chronic Wasting Disease.

Chronic wasting disease (CWD) is a contagious, fatal, neurodegenerative prion disease of cervids. The expanding geographical range and rising prevalence of CWD are increasing the risk of pathogen transfer and spillover of CWD to non-cervid sympatric species. As beavers have close contact with environmental and food sources of CWD infectivity, we hypothesized that they may be susceptible to CWD prions. We evaluated the susceptibility of beavers to prion diseases by challenging transgenic mice expressing beaver prion protein (tgBeaver) with five strains of CWD, four isolates of rodent-adapted prions and one strain of Creutzfeldt-Jakob disease. All CWD strains transmitted to the tgBeaver mice, with attack rates highest from moose CWD and the 116AG and H95+ strains of deer CWD. Mouse-, rat-, and especially hamster-adapted prions were also transmitted with complete attack rates and short incubation periods. We conclude that the beaver prion protein is an excellent substrate for sustaining prion replication and that beavers are at risk for CWD pathogen transfer and spillover.

Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer.

Chronic wasting disease (CWD) is a contagious and fatal transmissible spongiform encephalopathy affecting species of the cervidae family. CWD has an expanding geographic range and complex, poorly understood transmission mechanics. CWD is disproportionately prevalent in wild male mule deer and male white-tailed deer. Sex and species influences on CWD prevalence have been hypothesized to be related to animal behaviours that involve deer facial and body exocrine glands. Understanding CWD transmission potential requires a foundational knowledge of the cellular prion protein (PrPC) in glands associated with cervid behaviours. In this study, we characterized the presence and distribution of PrPC in six integumentary and two non-integumentary tissues of hunter-harvested mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus). We report that white-tailed deer expressed significantly more PrPC than their mule deer in the parotid, metatarsal, and interdigital glands. Females expressed more PrPC than males in the forehead and preorbital glands. The distribution of PrPC within the integumentary exocrine glands of the face and legs were localized to glandular cells, hair follicles, epidermis, and immune cell infiltrates. All tissues examined expressed sufficient quantities of PrPC to serve as possible sites of prion initial infection, propagation, and shedding.

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

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: The 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.

Metformin Treatment in Old Rats and Effects on Mitochondrial Integrity.

Metformin, a commonly used well-tolerated treatment for type 2 diabetes, is being deployed in clinical trials to ameliorate aging in older nondiabetic humans. Concerningly, some experiments in model organisms have suggested that metformin use at old ages shortens life span and is toxic to mitochondria. The demonstrated safety of metformin therapy in humans and the conflicting data from model organisms compelled us to test the hypothesis that metformin treatment would be toxic to older rats. To define an effective dose in 30-month-old hybrid rats, we evaluated two doses of metformin (0.1%, 0.75% of the diet) and treated the rats for 4 months. Body mass decreased at the 0.75% dose. Neither dose affected mortality between 30 and 34 months of age. We assessed mitochondrial integrity by measuring mitochondrial DNA (mtDNA) copy number and deletion mutation frequency, and mitochondrial respiration in skeletal muscle and the heart. In skeletal muscle, we observed no effect of metformin on quadriceps mass, mtDNA copy number, or deletion frequency. In the heart, metformin-treated rats had higher mtDNA copy number, lower cardiac mass, with no change in mtDNA deletion frequency. Metformin treatment resulted in lower mitochondrial complex I-dependent respiration in the heart. We found that, in old rats, metformin did not compromise mtDNA integrity, did not affect mortality, and may have cardiac benefits. These data provide some reassurance that a metformin intervention in aged mammals is not toxic at appropriate doses.

Chronic wasting disease: a cervid prion infection looming to spillover.

The spread of chronic wasting disease (CWD) during the last six decades has resulted in cervid populations of North America where CWD has become enzootic. This insidious disease has also been reported in wild and captive cervids from other continents, threatening ecosystems, livestock and public health. These CWD "hot zones" are particularly complex given the interplay between cervid PRNP genetics, the infection biology, the strain diversity of infectious prions and the long-term environmental persistence of infectivity, which hinder eradication efforts. Here, we review different aspects of CWD including transmission mechanisms, pathogenesis, epidemiology and assessment of interspecies infection. Further understanding of these aspects could help identify "control points" that could help reduce exposure for humans and livestock and decrease CWD spread between cervids.

New and distinct chronic wasting disease strains associated with cervid polymorphism at codon 116 of the Prnp gene.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Polymorphisms in the prion protein gene can result in extended survival of CWD-infected animals. However, the impact of polymorphisms on cellular prion protein (PrPC) and prion properties is less understood. Previously, we characterized the effects of a polymorphism at codon 116 (A>G) of the white-tailed deer (WTD) prion protein and determined that it destabilizes PrPC structure. Comparing CWD isolates from WTD expressing homozygous wild-type (116AA) or heterozygous (116AG) PrP, we found that 116AG-prions were conformationally less stable, more sensitive to proteases, with lower seeding activity in cell-free conversion and reduced infectivity. Here, we aimed to understand CWD strain emergence and adaptation. We show that the WTD-116AG isolate contains two different prion strains, distinguished by their host range, biochemical properties, and pathogenesis from WTD-116AA prions (Wisc-1). Serial passages of WTD-116AG prions in tg(CerPrP)1536+/+ mice overexpressing wild-type deer-PrPC revealed two populations of mice with short and long incubation periods, respectively, and remarkably prolonged clinical phase upon inoculation with WTD-116AG prions. Inoculation of serially diluted brain homogenates confirmed the presence of two strains in the 116AG isolate with distinct pathology in the brain. Interestingly, deglycosylation revealed proteinase K-resistant fragments with different electrophoretic mobility in both tg(CerPrP)1536+/+ mice and Syrian golden hamsters infected with WTD-116AG. Infection of tg60 mice expressing deer S96-PrP with 116AG, but not Wisc-1 prions induced clinical disease. On the contrary, bank voles resisted 116AG prions, but not Wisc-1 infection. Our data indicate that two strains co-existed in the WTD-116AG isolate, expanding the variety of CWD prion strains. We argue that the 116AG isolate does not contain Wisc-1 prions, indicating that the presence of 116G-PrPC diverted 116A-PrPC from adopting a Wisc-1 structure. This can have important implications for their possible distinct capacities to cross species barriers into both cervids and non-cervids.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

White-tailed deer S96 prion protein does not support stable in vitro propagation of most common CWD strains.

PrPC variation at residue 96 (G/S) plays an important role in the epidemiology of chronic wasting disease (CWD) in exposed white-tailed deer populations. In vivo studies have demonstrated the protective effect of serine at codon 96, which hinders the propagation of common CWD strains when expressed in homozygosis and increases the survival period of S96/wt heterozygous deer after challenge with CWD. Previous in vitro studies of the transmission barrier suggested that following a single amplification step, wt and S96 PrPC were equally susceptible to misfolding when seeded with various CWD prions. When we performed serial prion amplification in vitro using S96-PrPC, we observed a reduction in the efficiency of propagation with the Wisc-1 or CWD2 strains, suggesting these strains cannot stably template their conformations on this PrPC once the primary sequence has changed after the first round of replication. Our data shows the S96-PrPC polymorphism is detrimental to prion conversion of some CWD strains. These data suggests that deer homozygous for S96-PrPC may not sustain prion transmission as compared to a deer expressing G96-PrPC.