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Structural effects of PrP polymorphisms on intra- and inter-species prion transmission

Posted Aug 03 2014 2:20am
Structural effects of PrP polymorphisms on intra- and inter-species prion transmission

 

Rachel Angers2,5 Jeffrey Christansen1, Amy V. Nalls1, Hae-Eun Kang1, Nora Hunter3, Ed Hoover1, Candace K. Mathiason1, Michael Sheetz4 and Glenn C. Telling1,2,* 1 Prion Research Center (PRC) & the Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523 2 Department of Microbiology Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40506 3 The Roslin Institute and the University of Edinburgh, Midlothian, EH25 9RG 4 Center for Computational Sciences, University of Kentucky, Lexington, Kentucky 40506 5 Current address: N30 Pharmaceuticals, Inc., 3122 Sterling Circle, Suite 200, Boulder, Colorado 80301

 

Submitted to Proceedings of the National Academy of Sciences of the United States of America

 

Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra- and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling, and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cell-free systems. While Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared to glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between α helix 3 and the loop between β2 and α helix 2. These structure-function analyses are consistent with previous structural investigations, and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.

 

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This is the first report of which we are aware in which the effects of the white tailed deer PrP H95 and mule deer PrP F225 polymorphisms have been modeled in Tg mice. When assessing the results of incubation time experiments in Tg mice it is important to consider the effects of variations in transgene expression between lines. Frequently an inverse relationship exists between transgene expression and time to onset of disease (2), although this is not always the case (25). The Tg mice used here express deer, elk and deer-S96 PrP at five-fold greater than wild type, while levels are lower in Tg mice expressing deer- H95 and deer-F225 (Table S4), making it difficult to completely correlate variations in times to onset of disease among lines with the effects of polymorphic variation. Nonetheless, our studies on the susceptibility of Tg(DeerPrP-S96)7511+/- and Tg(DeerPrPH95) 7505+/- mice to CWD are consistent with the observed effects of experimentally infected deer expressing substitutions at positions 95 and 96 (23), and our results also confirm a protective role for F225 in CWD, previously suggested based on the rarity of the substitution in free-ranging deer with disease (7). Finally, the in vivo effects of polymorphisms on CWD and scrapie propagation were recapitulated in PMCA when levels of PrPC from the brains of various Tg mice were normalized to equivalent levels.

 

Previous studies consistently showed Tg60 mice expressing deer PrP-S96 to be entirely resistant to CWD, and investigators were unable to identify PrPSc in the brains of asymptomatic Tg60 mice (26, 27). Our results show that after long incubation times, CWD infected Tg(DeerPrP-S96)7511+/- mice were ultimately susceptible to disease, albeit with incomplete attack rates. Moreover, deer PrPSc-S96 was present in the brains of diseased as well as asymptomatic inoculated Tg(DeerPrP-S96)7511+/- mice, although at levels lower than deer PrPSc in the brains of diseased Tg(DeerPrP)1536+/- mice. The discrepancy is most likely related to the low transgene expression in Tg60 mice, reported to be 70% the levels found in deer. CWD occurs naturally in deer homozygous for the PrP-S96 allele (28), which is clearly inconsistent with a completely protective effect of this substitution, suggesting that Tg(DeerPrP-S96)7511+/- mice represent an accurate Tg model in which to assess the effects of the S96 substitution.

 

Finally, our findings showing that Tg(DeerPrP), but not Tg(ElkPrP) are sensitive to infection with SSBP/1 belie previously published results showing that SSBP/1 of the same provenance caused disease in two lines of Tg mice expressing elk PrP (13). However, our results appear to be consistent with the reported susceptibilities of elk and deer to sheep prions. In previous studies, of six elk inoculated with scrapie, three presented with neurological signs and neuropathology, but only after long and variable times to disease onset ranging from 25 to 46 months (29). In contrast, our results with SSBP/1 demonstrate relatively facile transmission of scrapie to deer, with all inoculated animals developing within 19 to 20 months, which is in accordance with susceptibility of deer to a US scrapie isolate with a similar time to disease onset (24). Polymorphisms ovine PrP add a further level of complexity, since they control the propagation scrapie strains. Occupancy of residue 136 by A or V is of particular importance. Our previous results indicated that SSBP/1 is comprised of a dominant strain that is preferentially propagated by sheep PrP encoding V at 136 (12). In contrast, the scrapie prions used in the deer transmission studies of Greenlee and colleagues were isolated from a sheep encoding A136, ***raising the possibility that deer may be susceptible to multiple scrapie strains.

 

Significance

 

The unpredictable recurrences of prion epidemics, their incurable lethality, and the capacity of animal prions to infect humans, provide significant motivation to ascertain the parameters governing disease transmission. The unprecedented spread, and uncertain zoonotic potential of chronic wasting disease (CWD), a contagious epidemic among deer, elk, and other cervids, is of particular concern. Here we demonstrate that naturally occurring primary structural differences in cervid PrPs differentially impact the efficiency of intra- and interspecies prion transmission. Our results not only deliver new information about the role of primary structural variation on prion susceptibility, but also provide functional support to a mechanism in which plasticity of a tertiary structural epitope governs prion protein conversion and intra- and inter-species susceptibility to prions.-

 

Reserved for Publication Footnotes

 

prions j protein structure j α helix 3/β2-α2 loop j transgenic mice

 


 

Virulence 4:4, 1–2; May 15, 2013; © 2013 Landes Bioscience

 

NEWS NEWS

 

Prion-resistant or prion-susceptible species, this is the question

 

Comment on: Chianini F, et al. Proc Natl Acad Sci U S A 2012; 109:5080-5; PMID:22416127;

 


 

Francesca Chianini,1 Natalia Fernández-Borges,2 Hasier Eraña,2 Yvonne Pang,2 Enric Vidal,3 Samantha L. Eaton,1 Jeanie Finlayson,1 Mark P. Dagleish1 and Joaquín Castilla2,4,*; 1Moredun Research Institute; Pentlands Science Park; Penicuik, Scotland UK; 2CIC bioGUNE; Parque tecnológico de Bizkaia; Bizkaia, Spain; 3Centre de Recerca en Sanitat Animal (CReSA); UAB-IRTA; Campus de la Universitat Autònoma de Barcelona; Barcelona, Spain; 4IKERBASQUE; Basque Foundation for Science; Bizkaia, Spain; *Email: castilla@joaquincastilla.com; http://dx.doi.org/10.4161/viru.24456

 

Previous in vivo studies left the scientific community with the assumption that rabbits were resistant to prion diseases. However, our recent findings proved they are susceptible. The in vitro results were essential to demonstrate that prion protein (PrP) from every species has the potential to become not only misfolded to a disease associated form, but also capable of being virulent and causing clinical disease. Even though transmissible spongiform encephalopathies have only been described in mammals to date, it would not be too surprising if prion diseases could eventually be found in any class of animal that has PrP such as birds, reptiles or fish.

 

The first reported observations of a transmissible spongiform encephalopathy (TSE) in Europe were in the first half of the 18th century when Thomas Comber described a disease of sheep, originally called rickets, which we know today as scrapie. However, the ability of TSEs to transmit to other species was unknown until 1960–1970 when the first experimental infections were performed in mice and other laboratory animals.

 

The differences between TSEs and other contagious diseases were evident early on, starting with the unusual characteristics of their pathogenesis, their unknown origin and especially their ability to be transmitted experimentally to a large number of species, even though different species are not equally susceptible (Barlow et al., Res Vet Sci 1976).

 

In the 1990s and early 21st century the main aim of TSE research was to establish the etiological agent, and although accomplishing this objective was of fundamental importance, it required most of the available funding resources and thereby prevented investigations of other aspects of these diseases.

 

Whereas confirmation of the “protein only hypothesis” represented a significant step forward for TSE science, it made the strain phenomenon and transmissibility between species more difficult to explain. Undoubtedly, it would have been easier to explain TSEs if the etiological agent was a virus or a bacterium, instead of one whose principal or only component is a protein.

 

In the past, many experimental infections were performed using different sources of TSEs, both within and between species, in an attempt to understand their pathogeneses and transmissibilities. However, in many occasions the existence of different strains was ignored.

 

The appearance of bovine spongiform encephalopathy (BSE) advanced knowledge in this area as a large number of animals were accidentally exposed to a novel TSE agent (Bons et al., Proc Natl Acad Sci U S A 1999). This “unplanned experiment” also showed that not every species was equally susceptible. For example, BSE was found in the goat population in the UK and France, but no cases were reported in pigs, despite proven experimental susceptibility, and having been naturally exposed to the agent during the BSE outbreak. However, we should not generalize with respect to susceptibility to prion diseases as their behaviors, and possibly even their mechanisms, can be as numerous as the number of identified strains. Without knowledge of the intrinsic characteristic of strains, the observation of natural and experimental infections may lead us to think that every strain is a unique and independent agent. This is because, despite having several similar characteristics, sometimes the different TSE strains behave as differently as the influenza virus does from the hepatitis C viruses. For example, different TSE strains target different species and tissues, with different incubation times and result in different clinical manifestations. Due to these innate differences, predicting if a strain will transmit to another species is very difficult and suggesting that a disease associated prion protein generated in cows either can or cannot transmit to humans is also dangerous. At best, we can try estimating the zoonotic potential of animal prions with ad hoc models such as primates or human PrP transgenic mice. Furthermore, it is currently impossible to establish if and how these unconventional agents will adapt and mutate when they infect new species. For all these reasons it is ill-advised to define a species as resistant to prion diseases on the basis of absence of natural cases or experiments where one can only use a limited number of strains.

 

The degree of pathogenicity of different disease associated prions, or virulence, is determined by the incidence of infection and the length of time between exposure and development of clinical signs. These data allow the classification of prion diseases from low to high virulence, but only if related to a specific species. This is because a TSE which is highly virulent in one species can be of low virulence or even avirulent in another. This paradigm of transmission is influenced by both the TSE strain and the species it is infecting, therefore, it is possible that every species has a specific strain that, once adapted, would represent the most virulent disease associated prion in that species.

 

The route of infection in TSEs plays a critical role in transmissibility and also the capacity of prions to replicate extraneurally is strainspecific (Beringue et al., Science 2012). This is often also responsible for the virulence of a strain in the same species. An exceptional example of transmissibility is scrapie, which, despite having been recognized for centuries as being highly virulent in sheep and goats, has never been reported as a natural infection in any other species and is therefore considered avirulent in humans. However, no one can be totally sure about its ability to adapt to other species. With respect to this it is important to mention that when BSE has been transmitted to sheep it becomes more virulent on re-passage as denoted by shorter incubation times in cattle and by an increase in the number of species it is capable of infecting (Padilla et al., PLoS Pathog 2011). These changes could happen with other TSE strains.

 

It is interesting to consider the potential virulence in common domestic species in which spontaneous prion diseases have never been reported. We should be very cautious in predicting the behavior of TSEs in these animals as transmissibility will depend on the combination of strain and challenged species. Nevertheless one should not consider this area of research an unanswerable enigma unless all strains are tested in every species, as this is unrealistic. To resolve this situation and start addressing some of these questions we have used our expertise in the in vitro replication of prions (Castilla et al., Cell 2005). We have examined a large number of TSE strains/ challenge species transmission combinations and performed a two passage study on the susceptibility of rabbits to in vitro generated homologous species disease associated prion infection (Chianini et al., Proc Natl Acad Sci U S A 2012). Prior to this study there were many uncertainties with respect to the susceptibility of rabbits to TSEs; previous in vivo studies had failed to transmit the disease yet the success of our in vitro studies proved that rabbit PrP could be efficiently misfolded after being seeded with different prion strains from different species and even formed an infectious de novo strain from unseeded brain.

 

The results of the in vivo studies left the scientific community with the assumption that rabbits were resistant to prion diseases. However, our recent findings proved they are susceptible. The in vitro results were essential to demonstrate that PrP from every species has the potential to become not only misfolded to a disease associated form, but also capable of being virulent and causing clinical disease. In our case rabbit PrP misfolded in vitro and produced a de novo proteaseresistant PrP from a healthy rabbit brain. This de novo PrP was capable of infecting a small percentage of rabbits on primary passage but a very high percentage succumbed to clinical disease upon second passage. Although with respect to our definition of virulence we could not consider our strain to be highly virulent; the mean for the incubation time was around 550 d post infection, we should not forget that several factors can influence the incubation time without altering the virulence. A clear example of this would be the long incubation times associated with human TSE strains in human infections, which are highly virulent.

 

From the production of the de novo TSE strain derived from the brain of a healthy rabbit it is tempting to speculate that its formation may be comparable to the spontaneous forms of prion disease, called atypical, and reported in humans and ruminants. These forms of prion diseases have always proven to be efficiently transmitted to the homologous species.

 

Even though we demonstrated that rabbits are not resistant to prion diseases, the studies performed in vivo previously and then confirmed with our study, showed that this species is not susceptible to TSE strains commonly virulent in other species such as ME7 in mice. These findings highlight the importance of the compatibility between the infectious PrP and the native PrP of the challenged species. This compatibility is dependent upon the amino acid sequence of the PrP and differences between the two proteins can determine the success or not of replication of the disease associated form.

 

Unfortunately, a simple comparison of PrP amino acid sequences between the species where strains have originated and the ones which are to be investigated cannot determine which PrP amino acids are responsible for successful disease transmission. This is not surprising since different strains with a different clinical course can be raised from the same species which has the same PrP amino acid sequence. To make things more complex, intermediate hosts can change the ability of certain prion diseases to become infectious in a species that otherwise appears not to be susceptible. The mechanism of how this happens is unclear, but the intermediate host may induce a conformational change or a mutation in the disease associated PrP strain or just aid the infectious ability of this strain in a different PrP environment. Therefore, thanks to intermediate hosts, certain TSE strains can increase their virulence and spread to other species as happened for BSE transmitted to sheep as previously explained.

 

In conclusion, even though TSEs have only been described in mammals to date, it would not be too surprising if, given the chance to evolve through intermediate hosts, prion diseases could eventually be found in any class of animal that has PrP such as birds, reptiles or fish.

 

Virulence

 

News & Views

 

Prion-resistant or prion-susceptible species, this is the question

 

Comment on: Chianini F, et al. Proc Natl Acad Sci U S A 2012; 109:5080-5; PMID:22416127; http://dx.doi.org/10.1073/pnas.1120076109 Francesca Chianini,1 Natalia Fernández-Borges,2 Hasier Eraña,2 Yvonne Pang,2 Enric Vidal,3 Samantha L. Eaton,1 Jeanie Finlayson,1 Mark P. Dagleish1 and Joaquín Castilla2,4,*; 1Moredun Research Institute; Pentlands Science Park; Penicuik, Scotland UK; 2CIC bioGUNE; Parque tecnológico de Bizkaia; Bizkaia, Spain; 3Centre de Recerca en Sanitat Animal (CReSA); UAB-IRTA; Campus de la Universitat Autònoma de Barcelona; Barcelona, Spain; 4IKERBASQUE; Basque Foundation for Science; Bizkaia, Spain; *Email: castilla@joaquincastilla.com; http://dx.doi.org/10.4161/viru.24456

 


 

P.126: Successful transmission of chronic wasting disease (CWD) into mice over-expressing bovine prion protein (TgSB3985)

 

Larisa Cervenakova,1 Christina J Sigurdson,2 Pedro Piccardo,3 Oksana Yakovleva,1 Irina Vasilyeva,1 Jorge de Castro,1 Paula Saá,1 and Anton Cervenak1 1American Red Cross, Holland Laboratory; Rockville, MD USA; 2University of California; San Diego, CA USA; 3Lab TSE/OBRR /CBER/FDA; Rockville, MD USA

 

Keywords: chronic wasting disease, transmission, transgenic mouse, bovine prion protein

 

Background. CWD is a disease affecting wild and farmraised cervids in North America. Epidemiological studies provide no evidence of CWD transmission to humans. Multiple attempts have failed to infect transgenic mice expressing human PRNP gene with CWD. The extremely low efficiency of PrPCWD to convert normal human PrPC in vitro provides additional evidence that transmission of CWD to humans cannot be easily achieved. However, a concern about the risk of CWD transmission to humans still exists. This study aimed to establish and characterize an experimental model of CWD in TgSB3985 mice with the following attempt of transmission to TgHu mice.

 

Materials and Methods. TgSB3985 mice and wild-type FVB/ NCrl mice were intracranially injected with 1% brain homogenate from a CWD-infected Tga20 mouse (CWD/Tga20). TgSB3985 and TgRM (over-expressing human PrP) were similarly injected with 5% brain homogenates from CWD-infected white-tailed deer (CWD/WTD) or elk (CWD/Elk). Animals were observed for clinical signs of neurological disease and were euthanized when moribund. Brains and spleens were removed from all mice for PrPCWD detection by Western blotting (WB). A histological analysis of brains from selected animals was performed: brains were scored for the severity of spongiform change, astrogliosis, and PrPCWD deposition in ten brain regions.

 

Results. Clinical presentation was consistent with TSE. More than 90% of TgSB3985 and wild-type mice infected with CWD/Tga20, tested positive for PrPres in the brain but only mice in the latter group carried PrPCWD in their spleens. We found evidence for co-existence or divergence of two CWD/ Tga20 strains based on biochemical and histological profiles. In TgSB3985 mice infected with CWD-elk or CWD-WTD, no animals tested positive for PrPCWD in the brain or in the spleen by WB. However, on neuropathological examination we found presence of amyloid plaques that stained positive for PrPCWD in three CWD/WTD- and two CWD/Elk-infected TgSB3985 mice. The neuropathologic profiles in CWD/WTD- and CWD/Elkinfected mice were similar but unique as compared to profiles of BSE, BSE-H or CWD/Tg20 agents propagated in TgSB3985 mice. None of CWD-infected TgRM mice tested positive for PrPCWD by WB or by immunohistochemical detection.

 

Conclusions. To our knowledge, this is the first established experimental model of CWD in TgSB3985. We found evidence for co-existence or divergence of two CWD strains adapted to Tga20 mice and their replication in TgSB3985 mice. Finally, we observed phenotypic differences between cervid-derived CWD and CWD/Tg20 strains upon propagation in TgSB3985 mice. Further studies are underway to characterize these strains.

 

P.89: Prions survive long-term burial in soil with some groundwater dissemination

 

Allister JA Smith,1 Karen Fernie,1 Ben Maddison,2 Keith Bishop,2 Kevin Gough,3 and Robert A Somerville1 1The Roslin Institute; University of Edinburgh; Edinburgh, UK; 2ADAS Biotechnology Group, University of Nottingham; Nottingham, UK; 3University of Nottingham; Nottingham, UK

 

An intrinsic property of prions is their extreme resistance to degradation. When they are deposited within the environment, whether from inappropriate disposal by man or from fallen diseased livestock, there is the potential to further propagate cases of disease for many years. It is evidenced that the spread of scrapie in sheep and chronic wasting disease in deer have occurred in this manner.

 

We mimicked such scenarios under large-scale field conditions to determine the extent to which TSE infectivity survives or disseminates in soil and soil water over five years. The mouse passaged BSE strain, 301V, was used to spike buried bovine heads, or was buried as an uncontained bolus in large soil-filled lysimeters. Two soils were examined, a free-draining sandy loam and a water-retentive clay loam.

 

Infectivity, determined by bioassay in mice, was recovered from all heads exhumed annually for 5 years from both soil types, with little reduction in the amount of infectivity over time. Small amounts of infectivity were found in soil samples immediately surrounding the heads but not in samples remote from them. Commensurate with this there was no evidence of significant lateral movement of infectivity from the bolus buried in a large soil mass. However large amounts of infectivity were recovered at the original bolus burial site in both soils. There was limited vertical upward movement of infectivity from the bolus buried in clay and downward movement from the bolus buried in sand perhaps reflecting the clay soils propensity to flood.

 

Throughout the course of the experiment rainwater particulate from several lysimeters was trapped on glass-fibre filters. Extracts from these filters were subject to serial PMCA (protein misfolding cyclic amplification) which was optimised using 301V-spiked samples and blinded controls. All positive and negative control samples were correctly determined. We have tested 44 samples from rainwater passed through the clay lysimeter filters, and found 9 positive samples, mainly from the initial 8 months of the experiment.

 

We conclude that TSE infectivity is likely to survive burial for long time periods with minimal loss of infectivity and limited movement from the original burial site. However PMCA results have shown that there is the potential for rainwater to elute TSErelated material from soil which could lead to the contamination of a wider area. These experiments reinforce the importance of risk assessment when disposing of TSE risk materials.

 

P.121: Efficient transmission of prion disease through environmental contamination

 

Sandra Pritzkow, Rodrigo Morales, and Claudio Soto Mitchell Center for Alzheimer’s disease and related Brain disorders; University of Texas Medical School at Houston; Hourston, TX USA

 

Chronic wasting disease (CWD) is a prion disorder effecting captive and free-ranging deer and elk. The efficient propagation suggests that horizontal transmission through contaminated environment may play an important role. It has been shown that infectious prions enter the environment through saliva, feces, urine, blood or placenta tissue from infected animals, as well as by carcasses from diseased animals and can stay infectious inside soil over several years.

 

82 Prion Volume 8 Supplement

 

We hypothesize that environmental components getting in contact with infectious prions can also play a role for the horizontal transmission of prion diseases. To study this issue, surfaces composed of various environmentally relevant materials were exposed to infectious prions and the attachment and retention of infectious material was studied in vitro and in vivo. We analyzed polypropylene, glass, stainless steel, wood, stone, aluminum, concrete and brass surfaces exposed to 263K-infected brain homogenate. For in vitro analyses, the material was incubated in serial dilutions of 263K-brain homogenate, washed thoroughly and analyzed for the presence of PrPSc by PMCA. The results show that even highly diluted PrPSc can bind efficiently to polypropylene, stainless steel, glass, wood and stone and propagate the conversion of normal prion protein. For in vivo experiments, hamsters were ic injected with implants incubated in 1% 263K-infected brain homogenate. Hamsters, inoculated with 263K-contaminated implants of all groups, developed typical signs of prion disease, whereas control animals inoculated with non-contaminated materials did not.

 

In addition, in order to study the transmission in a more natural setting, we exposed a group of hamster to habit in the presence of spheres composed of various materials that were pretreated with 263K prions. Many of the hamsters exposed to these contaminated materials developed typical signs of the disease that were confirmed by immunohistological and biochemical analyses.

 

These findings suggest that various surfaces can efficiently bind infectious prions and act as carriers of infectivity, suggesting that diverse elements in the environment may play an important role in horizontal prion transmission.

 

P.138: Phenotypic diversity in meadow vole (Microtus pennsylvanicus) prion diseases following challenge with chronic wasting disease isolates

 

Christopher J Johnson,1 Christina M Carlson,1,2 Jay R Schneider,1 Jamie K Wiepz,1 Crystal L Meyerett-Reid,3 Mark D Zabel,3 Joel A Pedersen,2 and Dennis M Heisey1 1USGS National Wildlife Health Center; Madison, WI USA; 2University of Wisconsin— Madison; Madison, WI USA; 3Colorado State University; Fort Collins, CO USA

 

Chronic wasting disease (CWD), a prion disease of cervids (deer, elk and moose), is spreading unchecked through large sections of North America. Transmission of CWD among cervids is especially facile and can occur through direct animal-toanimal contact and indirectly through contact with prions shed from infected animals. The disease transmission threat posed by CWD to other wildlife species remains unknown, but other species are inevitably exposed to CWD by consumption of infectious materials and through contact with environmental CWD contamination.

 

In this study, we investigated the transmission and adaptation of various white-tailed deer CWD isolates in the meadow vole (Microtus pennsylvanicus), a native North American rodent that is sympatric with current CWD epizootics that we have previously established is susceptible to CWD. We found that serial subpassage of CWD from white-tailed deer homozygous for glycine at position 96 (96GG) of the prion protein in meadow voles resulted in the selection of a single prion strain that was characterized by homogeneity in incubation period, abnormal prion protein (PrPTSE) glycoform ratio, lesion profile and PrPTSE deposition pattern. In contrast, passage of CWD from heterozygous 96GS genotype deer produced four unique disease phenotypes upon first passage. Subpassage of these types ultimately resulted in selection of a single strain by third passage that was distinct from the 96GG genotype CWD-derived strain.

 

We also establish that meadow voles are susceptible to CWD via peripheral challenge, albeit with lower attack rates and longer incubation periods. Interestingly, oral challenge of meadow voles with CWD resulted in subclinical infection in primary passage animals, but manifested as clinical prion disease upon subpassage.

 

Our data establish that meadow voles are permissive to CWD via peripheral exposure route, suggesting they could serve as an environmental reservoir for CWD. Additionally, our data are consistent with the hypothesis that at least two strains of CWD circulate in naturally-infected cervid populations and provide evidence that meadow voles are a useful tool for CWD strain typing.

 

P.141: Abundant prion shedding in CWD-infected deer revealed by Realtime conversion

 

Edward A Hoover,1 Davin M Henderson,1 Nathaniel D Denkers,1 Candace K Mathiason,1 Matteo Manca,2,3 and Byron Caughey2 1Prion Research Center, Colorado State University; Fort Collins, CO USA; 2Laboratory of Persistent Viral Diseases, NI AID; Hamilton, MT USA; 3Department of Biomedical Sciences, University of Cagliari; Monserrato, Italy

 

Background/Introduction. Chronic wasting disease (CWD) is unique among prion diseases in its efficient lateral transmission in nature. While the presence of infectious prions in body fluids and excreta of infected cervids has been demonstrated by bioassay, the dynamics, magnitude, and consequences of prion shedding remain unknown. The present studies were undertaken to determine the kinetics, duration, and magnitude of prion shedding in infected white-tailed deer.

 

Materials and Methods. Longitudinal samples were collected from white-tailed deer over a 2-year span after either oral (n=11)] aerosol (n = 6) CWD exposure. The assay protocol employed phosphotungstic acid precipitation of either whole saliva or the pelleted fraction of urine to seed recombinant Syrian hamster prion PrP substrate in RT-QuIC reactions. Prion seeding activity was assayed in 8 replicates of each sample employing thioflavin T detection in a 96-well plate-based fluorometer. Prion seeding reaction rate was determined by taking the inverse of the time at which samples exceeded a threshold of 5 standard deviations above the mean fluorescence of negative controls (1/time to threshold). Seeding activity was quantitated by comparing the realtime conversion reaction rate to a standard curve derived from a reference bioassayed brain pool homogenate from deer with terminal CWD.

 

Results. We analyzed >200 longitudinally collected, blinded, then randomized saliva and urine samples from 17 CWDinfected and 3 uninfected white-tailed deer. We detected prion shedding as early as 3 months post exposure and sustained thereafter throughout the disease course in both aerosol and orally exposed deer. The incidence of non-specific false positive results from >500 saliva and urine samples from negative control deer was 0.8%. By comparing real-time reaction rates for these body fluids to a bioassayed serially diluted brain control, we estimated that ≤1 ml of saliva or urine from pre-symptomatic infected deer constitutes a lethal infectious prion dose.

 

Conclusion. CWD prions are shed in saliva and urine of infected deer as early as 3 months post infection and throughout the subsequent >1.5 year course of infection. In current work we are examining the relationship of prionemia to excretion and the impact of excreted prion binding to surfaces and particulates in the environment.

 

Acknowledgments. Support: NIH-RO1-NS-061902; Morris Animal Foundation D12ZO-045

 

P.154: Urinary shedding of prions in Chronic Wasting Disease infected white-tailed deer

 

Nathaniel D Denkers,1 Davin M Henderson, 1 Candace K Mathiason,1 and Edward A Hoover1 1Prion Research Center, Department of Microbiology, Immunology, and Pathology, Colorado State University; Fort Collins, CO USA

 

Background/Introduction. Chronic wasting disease (CWD) is unique among prion diseases in its efficient lateral transmission in nature, yet the dynamics and magnitude of shedding and its immediate and long term consequences remain unknown. The present study was designed to determine the frequency and time span in which CWD prions are shed in urine from infected white-tailed deer using adapted real-time quaking-induced conversion (RT-QuIC) methodology.

 

Materials and Methods. Longitudinal urine samples were collected by free catch or catheterization over a 2-year period from oral-route infected [CWD+ (n = 11)] and aerosol-route-infected [CWD+ (n = 6); CWD- (n = 3)] white-tailed deer. High speed centrifugation pelleted material from 500 µl of urine was treated with sodium phosphotungstic acid (Na-PTA), resuspended in 0.05% SDS buffer, and used as seed in RT-QuIC assays employing recombinant Syrian hamster prion PrP substrate. Eight (8) replicates of each sample were run and prion seeding activity was recorded as thioflavin T binding fluorescence (480 nm emission) using a fluorimeter-shaker. Samples were considered positive if they crossed an established threshold (5 standard deviations above the negative mean fluorescence).

 

Results. In our oral-route inoculation studies, prion seeding activity has been demonstrated in urine collected at 6 months post-inoculation in 6 of 10 deer (11 of 80 replicates; 14%), and intermittently at later time points in all 11 CWD+ exposed deer. Our aerosol-route inoculation studies also showed prion seeding activity in urine collected at 6 months post-inoculation in 1 of 2 deer (3 of 16 replicates; 19%), and intermittently at later time points in 4 of 6 CWD+ exposed deer. Urine from sham-inoculated control deer and all baseline samples yielded 3 false-positive prion seeding activities (3 of 352 replicates; 0.8%).

 

Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC) are shed in urine of infected deer as early as 6 months post inoculation and throughout the subsequent disease course. Further studies are in progress refining the real-time urinary prion assay sensitivity and we are examining more closely the excretion time frame, magnitude, and sample variables in relationship to inoculation route and prionemia in naturally and experimentally CWD-infected cervids.

 

Acknowledgments. Support: NIH: RO1-NS-061902 and Morris Animal Foundation: D12ZO-045

 

P.158: Structurally and phenotypically different prions in CWD-infected white-tailed deer

 

Martin L Daus, Peter Lasch, and Michael Beekes Robert Koch-Institut; Berlin, Germany

 

Prions can exist as multiple strains within mammals. We could detect, for the first time, two distinct chronic wasting disease (CWD) isolates in white-tailed deer (WTD).

 

WTD had been challenged with CWD from either mule deer (MD) or WTD. Brain-derived prions from MD-infected WTD and WTD-infected WTD could be distinguished by biochemical, biophysical and biological methods. PK-mediated limited proteolysis at different pH-values indicated conformational differences between pathological prion proteins (PrPTSE) from MD-infected WTD and WTD-infected WTD. More specifically, Fouriertransform infrared microspectroscopy revealed secondary structure differences between highly purified PrPTSE extracts from MD-infected WTD and WTD-infected WTD. Different sedimentation velocities of PrPTSE in gradient centrifugations provided additional evidence for structure differences between prions from MD-infected WTD and WTD-infected WTD. Brain homogenate from WTD-infected WTD showed a substantially lower seeding activity on cellular prion protein (PrPC) of Syrian hamsters in protein misfolding cyclic amplification (PMCA) than its conformationally distinct counterpart from MD-infected WTD. When hamsters were intracerebrally inoculated with brain tissue from MD-infected WTD disease could be transmitted, which was not observed after similar inoculation with brain homogenate from WTD-infected WTD. In an ongoing macaque-study both CWD-isolates are currently being further tested for their transmissibility to primates.

 

P.163: Bayesian hierarchical modeling of chronic wasting disease in free-ranging white-tailed deer in the eastern U.S.

 

Tyler S Evans1 and W David Walter2 1Pennsylvania Cooperative Fish and Wildlife Research Unit; The Pennsylvania State University; University Park, PA USA; 2US Geological Survey; Pennsylvania Cooperative Fish and Wildlife Research Unit; The Pennsylvania State University; University Park, PA USA

 

Introduction. Chronic wasting disease (CWD) is a prion disease that affects both free-ranging and captive cervid populations. In the past 45 years, CWD has spread from a single region in Colorado to all bordering states, as well as Canada, the Midwest and the northeastern United States. In 2005, CWD was detected in the eastern U.S. in a free-ranging white-tailed deer (Odocoileus virginianus) killed by a vehicle in West Virginia followed by positives from Virginia, Maryland, and Pennsylvania. Although considerable information has been learned about CWD in wildlife from several areas of the U.S. and Canada, little information is available on spatial epidemiology of disease in the eastern U.S.

 

Materials and Methods. In order to develop a CWD surveillance plan for the region, we determined covariates and the best scale for analysis by exploring habitat use and estimating the mean size of home range for deer in the central Appalachian region (6 km2). We conducted Bayesian hierarchical modeling in WinBUGS on 24 a priori models using 11,320 free-ranging white-tailed deer (69 positive, 11,251 negative) that have been tested for CWD since 2005. Testing for CWD was conducted using standard protocols on a variety of tissues extracted from hunter-harvested deer that included retropharyngeal lymph nodes, tonsil lymph nodes, and the medulla oblongata sectioned at the obex.

 

Results. We found 94% of models weights were accounted for in our top model that identified habitats such as developed and open as covariates that increased the odds of infection for CWD in this region. Contrary to research in the endemic area of Colorado, we did not identify clay soil as a significant predictor of disease even though clay soil ranged from 9% to 19% in our study samples. Furthermore, contrary to results from the recent expansion of CWD into the agricultural Midwestern U.S. (Wisconsin, Illinois), we identified developed and open habitats were better predictors of disease occurrence compared to forest habitat considered more critical to deer population dynamics in the U.S.

 

Conclusions. Our results suggested that the odds of infection for CWD is likely controlled by areas that congregate deer thus increasing direct transmission (deer-to-deer interactions) or indirect transmission (deer-to-environment) by sharing or depositing infectious prion proteins in these preferred habitats. Epidemiology of CWD in the eastern U.S. is likely controlled by separate factors than found in the Midwestern and endemic areas for CWD and can assist in performing more efficient surveillance efforts for the region.

 

P.178: Longitudinal quantitative analysis of CWD prions shed in saliva of deer

 

Davin M Henderson, Nina Garbino, Nathaniel D Denkers, Amy V Nalls, Candace K Mathiason, and Edward A Hoover Prion Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University; Fort Collins, CO USA

 

Background/Introduction. Chronic Wasting Disease (CWD) is an emergent rapidly spreading fatal prion disease of cervids (deer, elk and moose). CWD has now been identified in 22 States (including two new states within the last year), 2 Canadian provinces, and South Korea. Shedding of infectious prions in excreta (saliva, urine, feces) may be an important factor in CWD transmission. Here we apply an adapted version of a rapid in vitro assay [real-time quaking-induced conversion (RT-QuIC)] to determine the time of onset, length, pattern, and magnitude of prion shedding in saliva of infected deer.

 

Materials and Methods. The RT-QuIC assay was performed as previously described in Henderson et al. PLoS-One (2013). Saliva samples were quantitated by comparison to a RT-QuIC reaction rate standard curve of a bioassayed obex sample from a terminally ill cervid.

 

Results. To better understand the onset and length of CWD prion shedding we analyzed >150 longitudinally collected, blinded, then randomized saliva samples from 17 CWD-infected and 3 uninfected white-tailed deer. We observed prion shedding, as detected by the RT-QuIC assay, as early as 3 months from inoculation and sustained shedding throughout the disease course in both aerosol and orally exposed deer. We estimated the infectious lethal dose of prions shed in saliva from infected deer by comparing real-time reaction rates of saliva samples to a bioassayed serially diluted brain control. Our results indicate that as little as 1 ml of saliva from pre-symptomatic infected deer constitutes a lethal CWD prion dose.

 

Conclusions. During the pre-symptomatic stage of CWD infection and throughout the course of disease deer may be shedding multiple LD50 doses per day in their saliva. CWD prion shedding through saliva and excreta may account for the unprecedented spread of this prion disease in nature.

 

Acknowledgments. Supported by NIH grant RO1-NS-061902 and grant D12ZO-045 from the Morris Animal Foundation.

 


 

PRION 2014 CONFERENCE

 

CHRONIC WASTING DISEASE CWD

 

A FEW FINDINGS ;

 

Conclusions. To our knowledge, this is the first established experimental model of CWD in TgSB3985. We found evidence for co-existence or divergence of two CWD strains adapted to Tga20 mice and their replication in TgSB3985 mice. Finally, we observed phenotypic differences between cervid-derived CWD and CWD/Tg20 strains upon propagation in TgSB3985 mice. Further studies are underway to characterize these strains.

 

We conclude that TSE infectivity is likely to survive burial for long time periods with minimal loss of infectivity and limited movement from the original burial site. However PMCA results have shown that there is the potential for rainwater to elute TSE related material from soil which could lead to the contamination of a wider area. These experiments reinforce the importance of risk assessment when disposing of TSE risk materials.

 

The results show that even highly diluted PrPSc can bind efficiently to polypropylene, stainless steel, glass, wood and stone and propagate the conversion of normal prion protein. For in vivo experiments, hamsters were ic injected with implants incubated in 1% 263K-infected brain homogenate. Hamsters, inoculated with 263K-contaminated implants of all groups, developed typical signs of prion disease, whereas control animals inoculated with non-contaminated materials did not.

 

Our data establish that meadow voles are permissive to CWD via peripheral exposure route, suggesting they could serve as an environmental reservoir for CWD. Additionally, our data are consistent with the hypothesis that at least two strains of CWD circulate in naturally-infected cervid populations and provide evidence that meadow voles are a useful tool for CWD strain typing.

 

Conclusion. CWD prions are shed in saliva and urine of infected deer as early as 3 months post infection and throughout the subsequent >1.5 year course of infection. In current work we are examining the relationship of prionemia to excretion and the impact of excreted prion binding to surfaces and particulates in the environment.

 

Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC) are shed in urine of infected deer as early as 6 months post inoculation and throughout the subsequent disease course. Further studies are in progress refining the real-time urinary prion assay sensitivity and we are examining more closely the excretion time frame, magnitude, and sample variables in relationship to inoculation route and prionemia in naturally and experimentally CWD-infected cervids.

 

Conclusions. Our results suggested that the odds of infection for CWD is likely controlled by areas that congregate deer thus increasing direct transmission (deer-to-deer interactions) or indirect transmission (deer-to-environment) by sharing or depositing infectious prion proteins in these preferred habitats. Epidemiology of CWD in the eastern U.S. is likely controlled by separate factors than found in the Midwestern and endemic areas for CWD and can assist in performing more efficient surveillance efforts for the region.

 

Conclusions. During the pre-symptomatic stage of CWD infection and throughout the course of disease deer may be shedding multiple LD50 doses per day in their saliva. CWD prion shedding through saliva and excreta may account for the unprecedented spread of this prion disease in nature.

 

see full text and more ;

 

Monday, June 23, 2014

 

*** PRION 2014 CHRONIC WASTING DISEASE CWD

 


 

Thursday, July 03, 2014

 

*** How Chronic Wasting Disease is affecting deer population and what’s the risk to humans and pets?

 


 

Tuesday, July 01, 2014

 

*** CHRONIC WASTING DISEASE CWD TSE PRION DISEASE, GAME FARMS, AND POTENTIAL RISK FACTORS THERE FROM

 


 

Sunday, July 13, 2014

 

Louisiana deer mystery unleashes litigation 6 does still missing from CWD index herd in Pennsylvania Great Escape

 


 

Tuesday, May 20, 2014

 

“Atypical” Chronic Wasting Disease in PRNP Genotype 225FF Mule Deer

 


 

Monday, June 23, 2014

 

PRION 2014 TYPICAL AND ATYPICAL BSE AND CJD REPORT UPDATES

 


 

Monday, July 28, 2014

 

*** Mitigating the Risk of Transmission and Environmental Contamination of Transmissible Spongiform Encephalopathies 2013 Annual Report

 


 

Transmissible Spongiform Encephalopathy TSE Prion Disease North America 2014

 

Transmissible Spongiform Encephalopathy TSE Prion Disease have now been discovered in a wide verity of species across North America. typical C-BSE, atypical L-type BASE BSE, atypical H-type BSE, atypical H-G BSE, of the bovine, typical and atypical Scrapie strains, in sheep and goats, with atypical Nor-98 Scrapie spreading coast to coast in about 5 years. Chronic Wasting Disease CWD in cervid is slowly spreading without any stopping it in Canada and the USA and now has mutated into many different strains. Transmissible Mink Encephalopathy TME outbreaks. These Transmissible Spongiform Encephalopathy TSE Prion Disease have been silently mutating and spreading in different species in North America for decades.

 

The USDA, FDA, et al have assured us of a robust Triple BSE TSE prion Firewall, of which we now know without a doubt, that it was nothing but ink on paper. Since the 1997 mad cow feed ban in the USA, literally tons and tons of banned mad cow feed has been put out into commerce, never to return, as late as December of 2013, serious, serious breaches in the FDA mad cow feed ban have been documented. The 2004 enhanced BSE surveillance program was so flawed, that one of the top TSE prion Scientist for the CDC, Dr. Paul Brown stated ; Brown, who is preparing a scientific paper based on the latest two mad cow cases to estimate the maximum number of infected cows that occurred in the United States, said he has "absolutely no confidence in USDA tests before one year ago" because of the agency's reluctance to retest the Texas cow that initially tested positive. see ; http://www.upi.com/Health_News/2006/03/15/Analysis-What-that-mad-cow-means/UPI-12841142465253/

 

The BSE surveillance and testing have also been proven to be flawed, and the GAO and OIG have both raised serious question as to just how flawed it has been (see GAO and OIG reports). North America has more documented TSE prion disease, in different documented species (excluding the Zoo BSE animals in the EU), then any other place on the Globe. This does not include the very likelihood that TSE prion disease in the domestic feline and canine have been exposed to high doses of the TSE prion disease vid pet food. To date, it’s still legal to include deer from cwd zone into pet food or deer food. Specified Risk Material i.e. SRM bans still being breach, as recently as just last month.

 

nvCJD or what they now call vCJD, another case documented in Texas last month, with very little information being released to the public on about this case? with still the same line of thought from federal officials, ‘it can’t happen here’, so another vCJD blamed on travel of a foreign animal disease from another country, while ignoring all the BSE TSE Prion risk factors we have here in the USA and Canada, and the time that this victim and others, do spend in the USA, and exposed to these risk factors, apparently do not count in any way with regard to risk factor. a flawed process of risk assessment.

 

sporadic CJD, along with new TSE prion disease in humans, of which the young are dying, of which long duration of illness from onset of symptoms to death have been documented, only to have a new name added to the pot of prion disease i.e. sporadic GSS, sporadic FFI, and or VPSPR. I only ponder how a familial type disease could be sporadic with no genetic link to any family member? when the USA is the only documented Country in the world to have documented two different cases of atypical H-type BSE, with one case being called atypical H-G BSE with the G meaning Genetic, with new science now showing that indeed atypical H-type BSE is very possible transmitted to cattle via oral transmission (Prion2014). sporadic CJD and VPSPR have been rising in Canada, USA, and the UK, with the same old excuse, better surveillance. You can only use that excuse for so many years, for so many decades, until one must conclude that CJD TSE prion cases are rising. a 48% incease in CJD in Canada is not just a blip or a reason of better surveillance, it is a mathematical rise in numbers. More and more we are seeing more humans exposed in various circumstance in the Hospital, Medical, Surgical arenas to the TSE Prion disease, and at the same time in North America, more and more humans are becoming exposed to the TSE prion disease via consumption of the TSE prion via deer and elk, cattle, sheep and goats, and for those that are exposed via or consumption, go on to further expose many others via the iatrogenic modes of transmission of the TSE prion disease i.e. friendly fire. I pondered this mode of transmission via the victims of sporadic FFI, sporadic GSS, could this be a iatrogenic event from someone sub-clinical with sFFI or sGSS ? what if?

 

Two decades have passed since Dr. Ironside first confirmed his first ten nvCJD victims in 1995. Ten years later, 2005, we had Dr. Gambetti and his first ten i.e. VPSPR in younger victims. now we know that indeed VPSPR is transmissible. yet all these TSE prion disease and victims in the USA and Canada are being pawned off as a spontaneous event, yet science has shown, the spontaneous theory has never been proven in any natural case of TSE prion disease, and scientist have warned, that they have now linked some sporadic CJD cases to atypical BSE, to atypical Scrapie, and to CWD, yet we don’t here about this in the public domain. We must make all human and animal TSE prion disease reportable in every age group, in ever state and internationally, we must have a serious re-evaluation and testing of the USA cattle herds, and we must ban interstate movement of all cervids. Any voluntary effort to do any of this will fail. Folks, we have let the industry run science far too long with regards to the TSE prion disease. While the industry and their lobbyist continues to funnel junk science to our decision policy makers, Rome burns. ...end

 

REFERENCES

 

Sunday, June 29, 2014

 

Transmissible Spongiform Encephalopathy TSE Prion Disease North America 2014

 


 

TSS
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