Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Temporal Characterization of Prion Shedding in Secreta of White-Tailed Deer in Longitudinal Study of Chronic Wasting Disease, United States.

Chronic wasting disease (CWD) affects cervids in North America, Asia, and Scandinavia. CWD is unique in its efficient spread, partially because of contact with infectious prions shed in secreta. To assess temporal profiles of CWD prion shedding, we collected saliva, urine, and feces from white-tailed deer for 66 months after exposure to low oral doses of CWD-positive brain tissue or saliva. We analyzed prion seeding activity by using modified amyloid amplification assays incorporating iron oxide bead extraction, which improved CWD detection and reduced false positives. CWD prions were detected in feces, urine, and saliva as early as 6 months postinfection. More frequent and consistent shedding was observed in deer homozygous for glycine at prion protein gene codon 96 than in deer expressing alternate genotypes. Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids.

Variation in the prion protein gene (PRNP) open reading frame sequence in French cervids.

The recent emergence of chronic wasting disease (CWD) in Europe has become a new public health risk for monitoring of wild and farmed cervids. This disease, due to prions, has proliferated in North America in a contagious manner. In several mammalian species, polymorphisms in the prion protein gene (PRNP) play a crucial role in the susceptibility to prions and their spread. To obtain a reliable picture of the distribution of PRNP polymorphisms in the two most common cervid species in France, we sequenced the open reading frame (ORF) of this gene in 2114 animals, 1116 roe deer (Capreolus capreolus) and 998 red deer (Cervus elaphus). Selection criteria such as historical origin, spatial distribution and sex ratio have been integrated to establish this sample collection. Except for one heterozygous animal with a non-synonymous mutation at codon 37 (G37A), all the 1116 French roe deer were monomorphic. Red deer showed greater variation with two non-synonymous substitutions (T98A; Q226E), three synonymous substitutions (codons 21, 78 and 136) and a new 24pb deletion (Δ69-77). We found significant regional variations between French regions in the frequency of the identified substitutions. After cloning of the PRNP ORF from animals presenting multiple non-synonymous polymorphisms, we identified six haplotypes and obtained a total of twelve genotypes. As in other European countries, we highlighted the apparent homogeneity of PRNP in the French roe deer and the existence of a greater diversity in the red deer. These results were in line with European phylogeographic studies on these two species.

Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. RESULTS: A total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10-2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10-3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. CONCLUSIONS: Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

Genetic modulation of CWD prion propagation in cervid PrP Drosophila.

Chronic wasting disease is a fatal prion condition of cervids such as deer, elk, moose and reindeer. Secretion and excretion of prion infectivity from North American cervids with this condition causes environmental contamination and subsequent efficient lateral transmission in free-ranging and farmed cervids. Variants of cervid PrP exist that affect host susceptibility to chronic wasting disease. Cervid breeding programmes aimed at increasing the frequency of PrP variants associated with resistance to chronic wasting disease may reduce the burden of this condition in animals and lower the risk of zoonotic disease. This strategy requires a relatively rapid and economically viable model system to characterise and support selection of prion disease-modifying cervid PrP variants. Here, we generated cervid PrP transgenic Drosophila to fulfil this purpose. We have generated Drosophila transgenic for S138 wild type cervid PrP, or the N138 variant associated with resistance to chronic wasting disease. We show that cervid PrP Drosophila accumulate bona fide prion infectivity after exposure to cervid prions. Furthermore, S138 and N138 PrP fly lines are susceptible to cervid prion isolates from either North America or Europe when assessed phenotypically by accelerated loss of locomotor ability or survival, or biochemically by accumulation of prion seeding activity. However, after exposure to European reindeer prions, N138 PrP Drosophila accumulated prion seeding activity with slower kinetics than the S138 fly line. These novel data show that prion susceptibility characteristics of cervid PrP variants are maintained when expressed in Drosophila, which highlights this novel invertebrate host in modelling chronic wasting disease.

Novel Prion Strain as Cause of Chronic Wasting Disease in a Moose, Finland.

Our previous studies using gene-targeted mouse models of chronic wasting disease (CWD) demonstrated that Norway and North America cervids are infected with distinct prion strains that respond differently to naturally occurring amino acid variation at residue 226 of the prion protein. Here we performed transmissions in gene-targeted mice to investigate the properties of prions causing newly emergent CWD in moose in Finland. Although CWD prions from Finland and Norway moose had comparable responses to primary structural differences at residue 226, other distinctive criteria, including transmission kinetics, patterns of neuronal degeneration, and conformational features of prions generated in the brains of diseased mice, demonstrated that the strain properties of Finland moose CWD prions are different from those previously characterized in Norway CWD. Our findings add to a growing body of evidence for a diverse portfolio of emergent strains in Nordic countries that are etiologically distinct from the comparatively consistent strain profile of North America CWD.

Longitudinal detection of prion shedding in nasal secretions of CWD-infected white-tailed deer.

Chronic wasting disease (CWD) is an emergent prion disease spreading in cervid populations in North America, South Korea and Scandinavia. Rapid detection of CWD prions shed by live animals using minimally invasive methods remains an important need. Previous studies in deer, elk and hamsters have demonstrated prion replication in the nasal olfactory mucosa, yet the temporal profile of CWD prion shedding in nasal secretions has not been well characterized. Here we report nasal prion shedding in 18 deer orally exposed to low doses of CWD prions and monitored longitudinally by several parameters. Serially collected nasal swabs were assayed for CWD prion seeding activity using iron oxide magnetic extraction and real-time quaking-induced conversion (IOME RT-QuIC). These findings were correlated with the results from longitudinal tonsil biopsies, terminal tissues and PRNP genotype. We detected nasal prion shedding 3-16 months after the first positive tonsil biopsy in ten of the 18 deer; detectable shedding persisted thereafter in nine of the ten animals. Surprisingly, nasal swabs were negative in eight deer, even though all were CWD-infected as determined by tonsil biopsies and terminal tissue assays. Nasal shedding was detected more often in deer that were homozygous for glycine at codon 96, and those that were near or demonstrating symptoms of clinical disease shed earlier and more frequently, irrespective of prion exposure dose. The results of this study demonstrate nasal shedding of CWD prions that can be detected using minimally invasive nasal swab sampling and RT-QuIC analysis.

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.

Inter- and intra-species conversion efficacies of Norwegian prion isolates estimated by serial protein misfolding cyclic amplification.

Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease.

Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP).

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for CWD.

Heterogeneity of pathological prion protein accumulation in the brain of moose (Alces alces) from Norway, Sweden and Finland with chronic wasting disease.

Prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. They are characterized by the conformational conversion of the cellular prion protein (PrPC) into the pathological prion protein (PrPSc). In 2016, chronic wasting disease (CWD) gained great importance at European level due to the first disease detection in a wild reindeer (Rangifer tarandus) in Norway. The subsequent intensive CWD surveillance launched in cervids resulted in the detection of CWD in moose (Alces alces), with 11 cases in Norway, 3 in Finland and 4 in Sweden. These moose cases differ considerably from CWD cases in North American and reindeer in Norway, as PrPSc was detectable in the brain but not in lymphoid tissues. These facts suggest the occurrence of a new type of CWD. Here, we show some immunohistochemical features that are clearly different from CWD cases in North American and Norwegian reindeer. Further, the different types of PrPSc deposits found among moose demonstrate strong variations between the cases, supporting the postulation that these cases could carry multiple strains of CWD.

Distribution of the misfolded isoform of the prion protein in peripheral tissues and spinal cord of Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring chronic wasting disease.

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy of cervids associated with the presence of a misfolded prion protein (PrPCWD). Progression of PrPCWD distribution has been described using immunohistochemistry and histologic changes in a single section of brain stem at the level of the obex resulting in scores from 0 (early) to 10 (terminal) in elk with naturally occurring CWD. Here we describe the spread and distribution of PrPCWD in peripheral tissues and spinal cord in 16 wild and 17 farmed Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring CWD and correlate these findings with obex scores. Spinal cord and approximately 110 peripheral tissues were collected, processed, stained with hematoxylin and eosin, and immunolabeled with the anti-prion protein monoclonal antibody F99/97.6.1. The medial retropharyngeal and tracheobronchial lymph nodes were the first tissues to accumulate PrPCWD, followed by other lymphoid tissues, myenteric plexus, spinal cord, and finally tissues outside of the lymphatic and neural systems. However, the only significant histological lesion observed was mild spongiform encephalopathy in the dorsal column of the lower spinal cord in elk with an obex score of ≥9. Initial exposure to CWD prions may be through the respiratory system and spread appears to occur primarily via the autonomic nervous system. Therefore, we suggest using obex scores as a proxy for stage of disease progression and verifying with key peripheral tissues.

Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America.

Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe.

Humanized Transgenic Mice Are Resistant to Chronic Wasting Disease Prions From Norwegian Reindeer and Moose.

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy or prion disease affecting cervids. In 2016, the first cases of CWD were reported in Europe in Norwegian wild reindeer and moose. The origin and zoonotic potential of these new prion isolates remain unknown. In this study to investigate zoonotic potential we inoculated brain tissue from CWD-infected Norwegian reindeer and moose into transgenic mice overexpressing human prion protein. After prolonged postinoculation survival periods no evidence for prion transmission was seen, suggesting that the zoonotic potential of these isolates is low.

Cell wall deficiency - an alternate bacterial lifestyle?

Historically, many species of bacteria have been reported to produce viable, cell wall deficient (CWD) variants. A variety of terms have been used to refer to CWD bacteria and a plethora of methods described in which to induce, cultivate and propagate them. In this review, we will examine the long history of scientific research on CWD bacteria examining the methods by which CWD bacteria are generated; the requirements for survival in a CWD state; the replicative processes within a CWD state; and the reversion of CWD bacteria into a walled state, or lack thereof. In doing so, we will present evidence that not all CWD variants are alike and that, at least in some cases, CWD variants arise through an adaptive lifestyle switch that enables them to live and thrive without a cell wall, often to avoid antimicrobial activity. Finally, the implications of CWD bacteria in recurring infections, tolerance to antibiotic therapy and antimicrobial resistance will be examined to illustrate the importance of greater understanding of the CWD bacteria in human health and disease.

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.

Modelling the role of predation on disease burdens of prey.

Research Highlight: Brandell, E. E., Cross, P. C., Smith, D. W., Rogers, W., Galloway, N. L., MacNulty, D. R., Stahler, D. R., Treanor, J. & Hudson, P. J. (2022). Examination of the interaction between age-specific predation and chronic disease in the Greater Yellowstone Ecosystem. Journal of Animal Ecology, 00, 1-12. https://doi.org/10.1111/1365-2656.13661. Predation can alter disease dynamics in prey. If predators select for infected individuals, they can reduce disease burdens. In other cases, predators can increase disease burdens via various mechanisms such as altered prey behaviour. The influence of predation on disease dynamics is a result of interactions among various traits of the predators, prey and the pathogen itself. For example, pathogens tend to vary with age and predators typically select for certain age classes. Thus, the overlap between ages selected by predators and those infected will likely contribute to any effects of predation on reducing disease burdens. In this paper, Brandell et al. (2022) develop a model to evaluate the predator cleansing effect given age-based variation in pathogens and predation. The model was developed for Chronic Wasting Disease (CWD) infections in deer and elk facing predation by cougars and grey wolves in the Greater Yellowstone Ecosystem. The results indicate that predators can reduce CWD outbreak size, especially if selecting for infected individuals. CWD is an always fatal disease and this work suggests that predators could reduce disease burdens in cervids. The model is also applicable to other systems and promises to further our understanding of the role of predation on disease in prey, as well as drive future empirical studies.

Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.

A retrospective analysis of hearing after cholesteatoma surgery: the bony obliteration tympanoplasty versus canal wall up and canal wall down without mastoid obliteration.

OBJECTIVE: To evaluate and compare the hearing outcome after the bony obliteration tympanoplasty (BOT), canal wall up (CWU) without mastoid obliteration and canal wall down (CWD) without mastoid obliteration in a large patient cohort. As the aeration of the middle ear is associated with hearing outcome, we hypothesized that the post-operative hearing after the BOT may be better compared to CWU and CWD without obliteration. METHODS: This is a retrospective cohort study on all adult patients who underwent the BOT, CWU without obliteration or CWD without obliteration for primary or revision cholesteatoma between January 2003 and March 2019 with audiological follow-up at our institution. Pre-operative, short-term post-operative and long-term post-operative hearing tests were analyzed and potential factors influencing post-operative hearing were assessed. RESULTS: 626 ears were included. We found no significant differences between the short-term and long-term post-operative audiometry. The pre-operative air-bone gap (ABG) was the factor with the largest effect size on change in air-bone gap (ABG) between pre- and post-operative. When stratifying for this factor along with the type of ossicular chain reconstruction to account for differences at baseline, no significant differences in post-operative ABG were found between BOT and non-obliteration CWU and CWD. CONCLUSION: In this large retrospective cohort study, we found no significant differences in post-operative ABG between the BOT and the non-obliteration CWU and CWD. A solid comparison of hearing between groups remains very challenging as hearing outcome seems to be dependent on many different factors. Hearing outcome seems to be no additional argument to choose for BOT over non-obliteration surgery.