Structural effects of PrP polymorphisms on intra- and inter-species prion transmission
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.
snip...
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
posted by Terry S. Singeltary Sr. at
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